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PubMed1989

Modelling of reference values for size at birth.

Data from the Swedish Medical Birth Registration, 1977-1981 were used to apply methods of constructing reference standards for size at birth. Using clinical information a 'healthy' sub-population was extracted. The conditional distributions of birthweight (BW) and birthlength (BL) for each week of Gestational age, and the conditional distribution of birthweight given birthlength were modelled using truncated Normal distributions, after making use of Box-Cox power transformations. Spline functions were then used in conjunction with a multiplicative method to obtain appropriate percentage point curves. Examples of this analysis are given.

Birth Weight↗
PubMedDate not supplied

Receptor binding assay for paralytic shellfish poisoning toxins: optimization and interlaboratory comparison.

A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter. Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument. Precision, accuracy, and sensitivity [limit of detection = 0.2 microg saxitoxin (STX) equiv/100 g shellfish tissue] of the modified protocol were equivalent to those of the original protocol. The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values. Comparison of STX reference standards obtained from the U.S. Food and Drug Administration and the National Research Council, Canada, showed no observable differences. This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.

Amphibian Proteins↗
PubMed1987

Modified phosphoribosylpyrophosphate (PRPP) radioenzymatic assay: increased sensitivity, technical simplification and new applications.

Phosphoribosylpyrophosphate in amounts as low as 25 pmol could be reliably and economically measured with a CO2-releasing radioenzymatic assay when appropriate technical modifications were introduced. The concentration of commercially available phosphoribosylpyrophosphate used for reference standards was ascertained by a method based on the utilization of phosphoribosylpyrophosphate by hypoxanthine catalyzed by hypoxanthine phosphoribosyltransferase from red blood cell lysates. The addition of inorganic phosphate increased intracellular phosphoribosylpyrophosphate levels in HL-60 cell lysates and can be used to amplify low levels of phosphoribosylpyrophosphate. This phosphoribosylpyrophosphate assay amplified by inorganic phosphate has been developed to assay perturbations in the purine biosynthetic nucleotide pathway in response to various chemotherapeutic agents, such as anti-folates, or as a result of folate deficiency.

Cell Line↗
PubMed1986

Electrothermal determination of aluminum in biological samples by atomic absorption spectroscopy.

The successful application of any analytical technique involves a balance between the performance characteristics of the method and the data requirements of the problem. As has been discussed, the electrothermal procedure for the determination of aluminum imposes some constraints on the clinical application of the methodology. These include detection limits in the same range as normal serum values, the possibility of interference from sample constituents, and the potential for contamination. In addition, reference standards are not currently available. Nevertheless, electrothermal procedures have received broad acceptance. This can be attributed to several factors. Electrothermal procedures tend to be inherently simple. Second, an understanding of the potential limitations of the technique has led to procedures that minimize these negative aspects and provide accurate measurements of aluminum in biological fluids. These factors, in conjunction with the reliability and relative ease of application of the technique, have led to an acceptance of electrothermal atomic absorption as the method of choice for the measurement of aluminum in biological samples.

Aluminum↗
PubMed1998

A novel immunohistochemical semiquantitative technique for endothelial constitutive nitric oxide synthase immunoreactivity in rat coronary artery.

It has been difficult to quantify protein production in small pathological specimens by conventional techniques. We describe a new method for semiquantification of immunohistochemical staining, which involves application of the enzyme-labeled avidin (LAB) technique, coupled with an ultra-sensitive and fast chemiluminescent substrate for alkaline phosphatase. The entire procedure can be completed in less than 3 hr. The final step involves X-ray film exposure for 30 min, and the optical density of the subsequent images is examined with a microcomputer imaging device. The optical densities are translated into relative protein concentrations by a reference standard curve, obtained via an immunoblot. To establish a model for semiquantification of endothelial constitutive nitric oxide synthase (eNOS) protein, we compared the coronary arteries of WKY rats fed a normal chow diet to the coronary arteries of WKY rats fed a cholesterol diet. Using this technique, we have found a relative 130-fold decrease in eNOS in the cholesterol-fed group compared to the normal chow-fed group.

Animals↗
PubMed1999

Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity.

Recent studies suggest that stool antigen assays are more sensitive and specific than microscopy for the diagnosis of Entamoeba histolytica infection. One hundred twelve patients presenting at 3 centers with symptoms or risk factors of E. histolytica infection were prospectively enrolled in this study to evaluate new diagnostic tests for infections with E. histolytica and Entamoeba dispar. Four ELISA-based stool antigen kits for detecting E. histolytica or E. dispar were blindly compared with stool microscopy. Amebic serology was assessed by indirect hemagglutination. When antigen assays were used as the reference standard, microscopy performed at referral centers was more specific (68.4% vs. 9.5%) but less sensitive (70.4% vs. 92.1%) than microscopy performed in community laboratories. Diagnosis with the E. histolytica test and Merlin Optimun S ELISA indicated that only 3 (4.2%) of 72 coproantigen-positive stools were positive for E. histolytica. Indirect hemagglutination was a good predictor of E. histolytica infection when titers of antibody to ameba were >/=1:512.

Animals↗
PubMedDate not supplied

Experimental evaluation of central venous pressure monitoring in the dog.

The measurement of central venous pressure (CVP) will provide objective information about intravascular blood volume. Limitations exist with direct and indirect methods of measuring CVP; however, the purpose of this study was to determine if such factors as catheter size, patient position, or measurements taken with either a water manometer or a strain-gauge transducer would affect the accuracy of CVP measurements. The study population consisted of six adult, mixed-breed dogs. Catheter sizes evaluated were 16- and 19-gauge. Patient positions evaluated were right and left lateral recumbencies. Right atrial pressures (RAPs) were used as the reference standards for CVP measurements. Measurements of RAP and CVP were obtained with a water manometer and a strain-gauge transducer. No significant differences were observed in measured CVPs for the effects of catheter size and right versus left lateral recumbency. A 2.7-mmHg difference in water manometry was demonstrated when compared to strain-gauge transducer measurements. Also, strain-gauge transducer measurements had less precision compared to water manometer measurements.

Animals↗
PubMed2005

Screening test accuracy studies: how valid are our conclusions? Application to visual inspection methods for cervical screening.

While the basic concepts associated with screening are simple, studying the value of new tests requires a very strict methodology. This paper summarizes lessons learned regarding appropriate methodologies to assess the value of new screening approaches using visual inspection with acetic acid (VIA), a screening test for cervical pre-cancerous lesions, as an example. In addition to being convenient to, safe for and acceptable by target community members, a screening test should be reliable and have good test characteristics (i.e. be able to discriminate well between early disease and non disease). Test reliability assesses the degree to which repeated measurements of the test yields the same result. To ensure reproducibility of study findings, test reliability should be assessed before any evaluation of test accuracy. The accuracy of a test (specificity and sensitivity) is measured using cross-sectional studies with adequate sample size. Several basic features are necessary to ensure internal validity for such studies: (a) final disease status data should be obtained for all subjects, (b) all tests results must be determined independently of previous results, (c) the reference standard used to determine the disease status should be accurate, (d) the full "spectrum" of the disease should be included in the study. The study results should also have external validity to be applicable to other populations to which the test will be applied. All these consideration are exemplified by 17 very heterogeneous studies published to date assessing VIA test accuracy. The assessment of a new screening test is the first step in researching a new cancer prevention strategy. For this reason, this step should be carefully addressed through rigorous studies.

Acetic Acid↗
PubMed2005

Quality assurance/quality control of foot and mouth disease solid phase competition enzyme-linked immunosorbent assay--Part I. Quality assurance: development of secondary and working standards.

International movement in animals and animal products has urged organisations like the World Organisation for Animal Health (OIE) to draw up guidelines to regulate and facilitate trade between Member Countries. However, as the global market continues to grow, further standardisation and harmonisation of antibody detection assays for infectious diseases are needed, especially regarding the development and use of reference materials. For OIE notifiable diseases for which primary or international reference standards are available or under development, National or Regional Reference Laboratories are encouraged to establish their own secondary and/or working standards. This paper describes the development of standards for the foot and mouth disease (FMD) solid phase competition enzyme-linked immunosorbent assay using positive serum obtained from calves vaccinated against the FMD virus. The procedure outlined in this manuscript can easily be extrapolated to similar serological assays and should lead to further international harmonisation of assays and test results.

Animals↗
PubMedDate not supplied

Elucidation of LSD in vitro metabolism by liquid chromatography and capillary electrophoresis coupled with tandem mass spectrometry.

The in vitro metabolism of D-lysergic acid diethylamide (LSD) by human liver microsomes was investigated. Tandem mass spectrometric techniques, using precursor and neutral loss scans, were employed in the initial search for drug metabolites. The determination of LSD human liver in vitro metabolites was performed by high-performance liquid chromatography and capillary electrophoresis coupled with tandem mass spectrometry. Two new in vitro metabolites, lysergic acid ethylamide (LAE) and 2-oxo-LSD, were positively identified; their structures were established by comparing with reference standards. Several other possible in vitro metabolites detected were suggested to be mono- and trioxylated metabolites of LSD. The major metabolic route of LSD by human liver microsomes is deethylation. Some results with LSD-positive human urine are presented. Among the LSD-related compounds detected in human urine specimens, iso-LSD was present at the highest concentration, followed by nor-LSD and isonor-LSD. Low concentrations of LAE and iso-LAE were also found in these urine specimens.

Chromatography, Liquid↗
PubMedDate not supplied

A concise synthesis of the 3-O-beta-D- and 4'-O-beta-D-glucuronide conjugates of trans-resveratrol.

trans-Resveratrol ((E)-3,4',5-trihydroxystilbene, 1) is a phytoalexin produced naturally in plants and grape skins as a stress metabolite protecting against fungal attack. Widespread interest in this apparently structurally simple molecule and synthetic stilbene analogues has arisen in recent years due to the discovery of its antioxidant, antiinflammatory, and anti-carcinogenic activities, among others. Although O-conjugation with glucuronic acid in vivo is known to represent a significant metabolic pathway for polyphenolic compounds in general and 1 in particular, preclinical studies have been hampered by the lack of chemically pure, completely characterized reference standards of both regioisomeric 3-O-beta-d- and 4'-O-beta-d-glucuronide conjugates of 1 for adequate identification and quantification of these significant metabolites. The present work describes a concise, convergent synthesis of both 3-O-beta-d- and 4'-O-beta-d-glucuronide conjugates of 1 using a strategy based on a novel Heck coupling of iodoaryl-O-beta-d-glucuronate esters with appropriately substituted styrenes, such that highly pure multimilligram to gram quantities of both the 3-O-beta-d- and 4'-O-beta-d-glucuronide conjugates of 1 can now be conveniently synthesized.

Glucuronides↗
PubMedDate not supplied

Evaluation of amino acid analysis as reference method to quantitate highly purified proteins.

The applicability of amino acid analysis for accurate quantitation of reference standard preparations of proteins has been evaluated. This approach is very useful since, in addition to absolute quantitative information, it also provides a measure of composition, partial identity, and purity in a single experiment. Comparisons with Kjeldahl nitrogen assay and/or UV measurements shows that amino acid analysis is reliable for the quantitation of small-to-medium size proteins in the molecular weight range of 6-22 kDa. For larger proteins such as immunoglobulins (150 kDa), amino acid analysis may "underestimate" the total protein concentration. These results also show the effect of recovery of individual residues on protein quantitation. As expected, the recovery of more than one stable residue could be used to calculate total protein content of samples, which is in good agreement with the results obtained by Kjeldahl nitrogen assay. However, the protein concentrations calculated from the total mass of the recovered residues appear to give relatively low estimates in almost all cases. Thus, it is concluded that amino acid analysis is an appropriate reference method only when stable residues are employed for quantitation, particularly for highly purified proteins of rDNA origin.

Amino Acids↗
PubMed2000

Detection of the plasma volume expander hydroxyethyl starch in human urine.

The plasma volume expander hydroxyethyl starch (HES) is usually administered in cases of hypovolaemic shocks but in 1998 the press reported its misuse in endurance sports. Since January 2000, it has been put on the list of prohibited substances of the International Olympic Committee (IOC) and its misuse is to ban by doping controls. Therefore, a rapid method enabling the screening for HES in human urine was developed which can be easily adopted by IOC laboratories to analyse routine urine samples for this remedy. Excretion study urine samples obtained from patients treated with HES, blank urine specimen and reference standards, were hydrolysed with hydrochloric acid and without any further purification of the resulting monosaccharides their per-timethylsilylated derivatives were performed. By means of gas chromatography-mass spectrometry the products were separated and the alpha- and beta-isomers of glucose, 2-, 3- and 6-hydroxyethyl glucose derivatives were identified. Typical ion traces of 2- and 3-substituted glucose (m/z 248, m/z 261 and m/z 235, m/z 248, respectively) support the fast determination of the substances whose electron impact mass spectra are presented and discussed.

Adult↗
PubMed2004

Confirmatory analysis for drugs of abuse in plasma and urine by high-performance liquid chromatography-tandem mass spectrometry with respect to criteria for compound identification.

Recently, high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) has become a powerful tool for quantitative confirmatory analysis of drugs of abuse and has begun to spread in the field of forensic toxicology. Guidelines for confirmatory analysis by GC/MS and LC/MS/MS have been published recently by several organizations (WADA, IOC, SOFT, GTFCh, EU). However, these guidelines have not yet been included in procedures for drug analysis with LC/MS/MS. The prerequisites for forensic confirmatory analysis by LC/MS/MS with respect to EU guidelines are chromatographic separation, a minimum number of two MS/MS transitions to obtain the required identification points and predefined thresholds for the variability of the relative intensities of the MS/MS transitions (MRM transitions) in samples and reference standards. LC/MS/MS methods for determination of several classes of drugs of abuse including some basic drugs (opiates, stimulants), cannabinoids and some of their phase-I- and phase-II-metabolites (especially glucuronides) in urine and serum of drug abusers and/or crime offenders or victims have been developed and validated following current recommendations and are presented in this paper. At least two MRM transitions for each substance were monitored to provide sufficient identification of drugs, deuterated analogues of analytes were used as internal standards for quantitation where possible and chromatographic separation has been performed on reversed-phase columns with gradient elution. Validation data obtained and the application to real samples show that the requested criteria for confirmatory analysis of drugs of abuse by EU guidelines can be fulfilled with a total number of four identification points by LC/MS/MS methods using a triple-quadrupole mass spectrometer. Furthermore, the methods are sufficiently sensitive to meet current requirements for confirmatory analysis of drugs of abuse in driving under the influence of drugs (DUID) cases established by the Society of Toxicological and Forensic Chemistry (GTFCh).

Chromatography, High Pressure Liquid↗
PubMed2003

Modified clean-up for PBDD, PBDF and PBDE with an active carbon column--its application to sediments.

A clean-up method for polybrominated dibenzo-p-dioxins (PBDDs), dibenzofurans (PBDFs) and diphenyl ethers (PBDEs) was modified using combinations of multi-layered silica gel, Florisil and active carbon columns. By using active carbon column chromatography in the final procedure, PBDEs were well separated from PBDD/Fs in an elution test with reference standards. We report the application of this method to sediment samples taken from industrialized areas in Japan. These sediments contained PBDEs ranging in concentration from 13 to 2394 pg/g, dry wt. PBDEs did not interfere with the quantification of PBDD/Fs. In addition, PBDEs found in the PBDD/F fraction did not interfere with the identification of PBDFs using the HRGC/HRMS separation method. Some 2,3,7,8-tetra- to hexabrominated dioxins and furans were also detected in the sediment samples.

Benzofurans↗
PubMedDate not supplied

Mineral content of Chinese medicinal herbs used as diuretic treatments for Taiwanese children.

Eighteen major, minor and trace elements in 12 Chinese medicinal herbs commonly consumed by Taiwanese Children as diuretics were determined by instrumental neutron activation analysis (INAA). Dried and powdered herb samples were irradiated in a neutron flux of ca. 2 x 10(12) n/cm2 s under separate short and long irradiation schemes. Lichen (IAEA-336) was used as the reference standard, and tomato leaves (NIST-SRM 1570a) were employed for cross-checking the accuracy of the results. INAA was shown to be a reliable multi-element analytical method for determining the content of both toxicologically and nutritionally important minerals in Chinese medicinal herbs. Determined elements were present in the dried herbs in concentrations ranging from 10(4) to 10(-3) microg/g. The mineral contents and the maximum daily intake values of the tested herbs were compared with published values and with the recommended daily intakes for Taiwanese children as specified by the World Health Organization.

Child↗
PubMedDate not supplied

Chemical synthesis and characterization of conjugates of a novel catechol-O-methyltransferase inhibitor.

BIA 3-202, 1-(3,4-dihydroxy-5-nitrophenyl)-2-phenylethanone 3, is a novel, reversible, and tight-binding peripheral inhibitor of the enzyme catechol-O-methyltransferase (COMT), which is currently under clinical evaluation for the treatment of Parkinson's disease as an adjunct to current L-Dopa/peripheral decarboxylase inhibitor therapy. Chemically pure, well-characterized reference standards of conjugates of 3 were required for investigation of the routes of metabolism in several animal species (including humans) and for pharmacokinetic studies. The lack of suitable literature precedents for efficient, regioselective synthesis of nitrocatechol conjugate metabolites prompted us to develop efficient and highly selective chemical preparations of O-glucuronide and O-sulfate conjugates of 3 such that multigram quantities of excellent purity can now be conveniently synthesized. It is anticipated that these procedures could be applied to the synthesis of conjugates of other COMT inhibitors, also based on the 3-nitrocatechol pharmacophore.

Acetophenones↗
PubMed1992

Organomercury determination in biological reference materials: application to a study on mercury speciation in marine mammals off the Faröe Islands.

The potential use of graphite furnace atomic absorption spectrometry (GF-AAS) for the organic mercury determination in marine biological tissues was evaluated. Following its isolation by acid extraction in toluene, organic mercury was recovered in aqueous thiosulfate and measured by GF-AAS. The detection limit was 0.01 microgram Hg/g (as methyl mercury). Analyses were conducted on three reference standard materials certified for their methyl mercury content, DOLT-1, DORM-1, and TORT-1, provided by the National Research Council of Canada. The method resulted in very good recovery and reproducibility, indicating that GF-AAS can provide results comparable to those obtained by using more expensive and time consuming analytical techniques. The method was applied to the analysis of liver tissues of pilot whale specimens (Globicephala melas) from the drive fishery of the Faröe Islands (northeast Atlantic). The results provided useful information on the proportion of different mercury forms in the liver of these marine mammals.

Animals↗