210Pb and 210Po in Alaskan biological samples. BNWL-235 2.
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Alaska↗
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A method for analyzing 233U in biological samples.
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Animals↗
Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples.
An amino acid analysis by reversed-phase high-performance liquid chromatography after precolumn derivatization with phenyl isothiocyanate was adapted to the determination of free amino acids in plasma or other biological fluids and in tissue homogenates. Preparation of samples included deproteinization by 3% sulphosalicylic acid, and careful removal under high vacuum of residual phenyl isothiocyanate after derivatization. A Waters Pico-Tag column (15 cm long) was used, immersed in a water-bath at 38 degrees C. In rat or human plasma, separation of 23 individual amino acids, plus the unresolved pair tryptophan and ornithine, was obtained within 13 min. Including the time for column washing and re-equilibration, samples could be chromatographed at 23-min intervals. Variability was tested for each amino acid by calculating the coefficients of variation of retention times (less than 1% in the average) and peak areas (less than 4% for both intra-day and inter-day determinations). The linearity for each standard amino acid was remarkable over the concentration range 3-50 nmol/ml. The mean recovery of amino acid standards added to plasma prior to derivatization was 97 +/- 0.8%, except for aspartate (82%) and glutamate (81%). This method is rapid (almost three samples per hour can be analysed, more than in any other reported technique), with satisfactory precision, sensitivity and reproducibility. Therefore, it is well suited for routine analysis of free amino acids in both clinical and research work.
Amino Acids↗
Sensitive high-performance liquid chromatography method for the determination of low levels of perchlorate in biological samples.
A rapid and sensitive high-performance liquid chromatography (HPLC) method was developed to detect perchlorate in tissues of male and female rats, both pregnant and lactating (including milk) after administration of perchlorate. Supernatants of ethanol precipitated rat fluids and tissues were evaporated to dryness under nitrogen and reconstituted in deionized water. Reconstituted samples were injected into HPLC system coupled with conductivity detection. Isocratic separation of perchlorate was achieved using an anion-exchange column with sodium hydroxide as mobile phase and a conductivity detector. In this method, perchlorate showed a linear response range from 5 to 100 ng/ml. The lower detection limits for perchlorate in fluids and tissues of rats were 3-6 ng/ml and 0.007-0.7 mg/kg, respectively. The described method has the unique advantage over the existing methods of determining low traces of perchlorate in different biological matrices without complex sample preparation.
Animals↗
The use of reference materials in the elemental analysis of biological samples.
Reference materials (RMs) are useful to compare the accuracy and precision of laboratories and techniques. The desirable properties of biological reference materials are listed, and the problems of production, homogenization and storage described. At present there are only 10 biological RMs available compared with 213 geological and 520 metallurgical RMs. There is a need for more biological RMs including special materials for microprobe analysis and for in vivo activation analysis. A study of 650 mean values for elements in RM Kale, analysed by many laboratories, leads to the following conclusions. 61% of the values lie within +/- 10% of the best mean, and 80% lie within +/- 20% of the best mean. Atomic absorption spectrometry gives results that are 5-30% high for seven elements, while instrumental neutron activation analysis gives low and imprecise results for K. Other techniques with poor interlaboratory precision include neutron activation for Mg, polarography for Zn and arc-spectrometry for many elements. More than half the values for elements in Kale were obtained by neutron activation, confirming the importance of this technique and the need for RMs. As a rough estimate, 6 X 10(9) elemental analyses of biological materials are carried out each year, mostly by medical, agricultural and food scientists. It seems likely that a substantial percentage of these are inaccurate, a situation that might be improved by quality control using standard RMs.
Chemistry Techniques, Analytical↗
Sensitive and rapid colorimetric immunoenzymometric assay of ferritin in biological samples.
We describe a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for quantifying ferritin in human and rat biological fluids. We used chlorophenol red beta-D-galactopyranoside as the colorimetric substrate of beta-galactosidase (EC 3.2.1.23), which is coupled to specific antibodies to either human or rat liver ferritin. The assay is sensitive (detection limit for human assay = 0.58 micrograms/L and for rat assay = 0.37 micrograms/L), accurate (average recovery for human assay = 93% and for rat assay = 92%), and precise (total CVs for human assay = 2.3-12.2% and for rat assay = 5.6-11.3%). The results correlated well with those of an established immunoradiometric technique (r = 0.99691). This assay has a prolonged shelf-life, is inexpensive, and utilizes a stable colorimetric substrate that requires relatively short incubation.
Animals↗
Determination of beta2-microglobulin in biological samples using an immunoenzymometric assay (chemiluminescence detection) or an immunoturbidimetric assay: comparison with a radioimmunoassay.
Monitoring beta2-microglobulin (beta2M) in biological fluids has gained considerable interest in pathologies such as haematologic malignancies, renal diseases, and chronic inflammatory diseases. Due to limitations of the RIA in the routine laboratory, we measure beta2M with non-isotopic methods. 189 patients suffering from myeloma (n=66), end stage renal failure (n=54) or inflammation (n=69) were included in this study. beta2M was determined in serum, urine and dialysate using an immunoenzymometric assay with chemiluminescence detection [Immulite Diagnostic Products Corporation (DPC), La Garenne Colombes, France] and an immunoturbidimetric assay (Olympus, Rungis, France). The data were compared with a radioimmunoassay (Immunotech, Marseille, France) taken as a reference. Using serum samples, the immunoenzymometric assay with chemiluminescence detection and the immunoturbidimetric assay have reliable analytical performances. Values obtained with serum samples are highly correlated with the radioimmunoassay (DPC/RIA r2=0.84; Olympus/RIA r2=0.94) whatever the type of pathology; however an over-estimation which could be related to cross reactivity with beta2M fragments was observed with the RIA method as suggested by crossover calibration and recovery studies. Values obtained with urinary samples (n=96) are closely related to those obtained with the RIA (DPC/RIA r2 = 0.98; Olympus/RIA: r2=0.99). Despite the low levels observed in dialysate (n=57) good correlations were observed between Olympus vs DPC (r2=0.85). By contrast, the two non-isotope methods are poorly related with the RIA method (DPC vs RIA r2=0.47 and Olympus vs RIA r2=0.54). In conclusion, the immunoenzymometric assay with chemiluminescence detection or the immunoturbidimetric assay could be used in the routine laboratory in order to determine beta2M in plasma, urine and dialysate.
Body Fluids↗
Separation of acylcarnitines from biological samples using high-performance liquid chromatography.
A reverse-phase high-performance liquid chromatography technique to separate carnitine and acylcarnitines from a biological matrix is described. The method utilizes a step gradient to provide baseline resolution of acylcarnitines (individually or by class) for subsequent quantification using a sensitive radioenzymatic assay. The method requires minimal sample preparation and prevents any contamination among groups of acylcarnitines. This technique has been applied to liver tissues of rats obtained under a variety of conditions. These studies demonstrate the validity and utility of the HPLC method while confirming the applicability of the perchloric acid fractionation of acylcarnitines by functional class. The present HPLC method permits resolution of long-chain acylcarnitines in the presence of large excess concentrations of carnitine and short-chain acylcarnitines (coelution of unesterified carnitine with long-chain acylcarnitines less than or equal to 0.05%). Thus, the method will be of use in the study of acylcarnitines in biological systems over a broad spectrum of metabolic conditions.
Animals↗
Storage of biological samples.
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Blood Preservation↗
Damage to biological samples caused by the electron beam during electron microscopy.
A new method has been developed which measures directly the beam damage suffered by biological specimens in the electron microscope. This method involves the use of radioautography to measure specific radioactivity of labeled specimens, either exposed or unexposed to the beam. Using this technique, it has been found that macromolecular samples such as ribosomes and R17 virions are severely damaged during standard electron microscopic operations: from 15 to 40% of the mass of the sample may be lost in a 30 sec exposure to the beam.
Autoradiography↗
Preparation of biological samples for transmission X-ray microanalysis: a review of alternative procedures to the use of sectioned material.
Although transmission X-ray microanalysis of biological material has traditionally been carried out mainly on sectioned preparations, a number of alternative procedures exist. These are considered under three major headings - whole cell preparations, analysis of cell homogenates and biological fluids, and applications of the technique to microsamples of purified biochemicals. These three aspects provide a continuous range of investigative level - from the cellular to the molecular. The use of X-ray microanalysis with whole cell preparations is considered in reference to eukaryote (animal) cells and prokaryotes - where it has particular potential in environmental studies on bacteria. In the case of cell homogenates and biological fluids, the technique has been used mainly with microdroplets of animal material. The use of X-ray microanalysis with purified biochemicals is considered in relation to both particulate and non-particulate samples. In the latter category, the application of this technique for analysis of thin films of metalloprotein is particularly emphasised. It is concluded that wider use could be made of the range of preparative techniques available - both within a particular investigation, and in diverse fields of study. Transmission X-ray microanalysis has implications for environmental, physiological and molecular biology as well as cell biology.
Animals↗
Replica exchange with solute tempering: a method for sampling biological systems in explicit water.
An innovative replica exchange (parallel tempering) method called replica exchange with solute tempering (REST) for the efficient sampling of aqueous protein solutions is presented here. The method bypasses the poor scaling with system size of standard replica exchange and thus reduces the number of replicas (parallel processes) that must be used. This reduction is accomplished by deforming the Hamiltonian function for each replica in such a way that the acceptance probability for the exchange of replica configurations does not depend on the number of explicit water molecules in the system. For proof of concept, REST is compared with standard replica exchange for an alanine dipeptide molecule in water. The comparisons confirm that REST greatly reduces the number of CPUs required by regular replica exchange and increases the sampling efficiency. This method reduces the CPU time required for calculating thermodynamic averages and for the ab initio folding of proteins in explicit water.
Algorithms↗
Thin-layer chromatographic determination of catecholamines, 5-hydroxytryptamine, and their metabolites in biological samples: a review.
A review of methodology for separation, detection, and quantitative determination of catecholamines, 5-hydroxytryptamine, and their acidic metabolites in biological tissue and fluids by thin-layer chromatography is presented. Selected procedures, including fluorometric scanning densitometry for catecholamine acetyl derivatives and color scanning densitometry for acids, are described.
Biogenic Amines↗
Analysis of free porphyrins in biological samples by high performance liquid chromatography.
High performance liquid chromatography has been used to separate and analyse the different free porphyrins in biological materials. The selective determination of uroporphyrin, coproporphyrin, protoporphyrin and hepta, hexa, pentacarboxylic porphyrins, the sensitive and quantitative fluorescence detection contribute to the clinical diagnosis of porphyria.
Chromatography, High Pressure Liquid↗
[Quantitative method of 5-fluorouracil and its metabolites in biological samples using high performance liquid chromatography].
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Animals↗
Whole-field fluorescence microscope with digital micromirror device: imaging of biological samples.
We have developed a whole-field fluorescence microscope equipped with a Digital Micromirror Device to acquire optically sectioned images by using the fringe-projection technique and the phase-shift method. This system allows free control of optical sectioning strength through computer-controlled alteration of the fringe period projected onto a sample. We have employed this system to image viable cells expressing fluorescent proteins and discussed its biological applications.
Calcium↗
Integrated approach to the multidimensional analysis of complex biological samples by microseparation techniques. Analysis of glycoprotein factor associated with cancer cachexia.
Microanalytical separation techniques including capillary liquid chromatography, capillary electrophoresis and capillary electrochromatography are suitable for detection of diagnostically important changes in the metabolic profiles of biological fluids. A prototype instrument was employed to serve as an integrated platform for the analysis of urine sample from patients suffering from cancer cachexia. The instrument provides for convenient, rapid and efficient multidimensional approach towards method development which would facilitate simultaneous analysis of complex biological mixtures by the above techniques.
Cachexia↗
Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry. II. Rapid survey method for profiling trace elements in body fluids.
A rapid survey of the elements in biological materials, covering most of the elements in the periodic table, is possible by using available software for semi-quantitative analysis (SEMI-QUANT) by inductively coupled plasma-mass spectrometry. The procedure takes 5 min after sample preparation and gives results with a precision (CV) of approximately 20%. At a 10-fold dilution, 13 elements can be consistently and reliably detected in serum and 15 elements in whole-blood samples. At present the most important limitation of this method is mass overlap by polyatomic species for some elements of interest (e.g., Cr, Mn, and V). However, for the set of elements that can be reliably determined at endogenous concentrations, including Li, B, Mg, Fe, Cu, Zn, Rb, and Sr, the rapid scanning capability may be useful. Although matrix effects limit the direct interpretation of the semi-quantitative output, reasonable estimates of concentration are attainable by using matrix-matched standards or by adding a multielement standard to an aliquot from one sample in the set. We also present an example of determination of 25 elements in saliva from a patient with extensive dental work: Components of many of his dental alloys were readily identified. The method may also prove useful for screening multiple toxic exposures to heavier elements, such as Pb, Tl, Cd, and Hg.
Adult↗