Elastic properties of cancellous bone: measurement by an ultrasonic technique

The high degree of porosity of cancellous bone makes elastic property measurement difficult by traditional mechanical testing methods. An ultrasonic technique is described with which mechanical properties of anisotropic, rigid, porous materials, such as cancellous bone, can be measured. The technique utilizes unique piezoelectric transducers operated in a continuous wave mode at a frequency of approximately 50 kHz. Both longitudinal and shear waves can be propagated and received with the transducers allowing both Young's moduli and shear moduli to be determined with the technique. A comparison between moduli measured with the ultrasonic technique and moduli measured with traditional mechanical testing shows the new method to be quite accurate in elastic property determination, (r2 = 0.935, Emech = 1.00E1dt + 23.3 MPa) (r2 = 0.656, Gmech = 1.08 Gult--3.3MPa).     

Measurement of inorganic pyrophosphate in biological fluids and bone tissues

A simple and rapid technique of measuring pyrophosphates in plasma, urine, and bone tissue is described, using the UDPG-pyrophosphorylase reaction and fluorimetrical determination of NADPH formed in a combined system of phosphorylation and reduction. This very specific method avoids the separation of Pi² by column chromatography, and its very great sensitivity enables measurement on a final sample corresponding to 0.2 ml of plasma, with a precision of about 3%.The mean plasma PPi concentration (±SE of mean) was 3.53 ± 0.19 μmoles/liter (SE=0.93), or 0.207 ± 0.006 mg Pi/liter. The normal range for this population (99% confidence limit) is therefore between 1.10 and 5.90 μmoles/liter or 0.068 – 0.366 mg Pi/liter. Analysis of the variation of the duplicate measurements shows a very small divergence of 3.4% or ±0.12 μm.Normal 24 hr urinary excretion is 53.0 ± 6.8 μmoles with 99% confidence limits of 10.0 and 96.0 μmoles.The average PPi concentration in calvaria of 20 10-day-old rats is 3.685 ± 0.16 nmoles/mg fresh weight.     

Extraction and measurement of deuterium oxide at tracer levels in biological fluids

Methodologies were developed which provided resolution of deuterium oxide in water and sublimates of biological fluids to ±1 ppm over the range of background to 1000 ppm added D₂O. These procedures involve a simple rapid sublimation procedure with infrared analyses, at 4.0 μm, using a simple Wilks infrared analyzer and 0.2-mm calcium fluoride cells with cell temperature regulated to ±0.005°C.     

Noncompetitive enzyme immunoassay for the measurement of bronchial inhibitor in biological fluids

An enzyme-linked immunosorbent assay (ELISA) of bronchial inhibitor using rabbit antibronchial inhibitor antibody-coated polystyrene balls as the solid-phase antibody and peroxidase-labeled antibody as the conjugate is described. A crude antibody fraction is used for coating the solid phase. The assay can be run within 8 h and gives reproducible results in the range of 6 to 60 micrograms/l of bronchial inhibitor (mean within-run coefficient of variation, 7%). It can detect bronchial inhibitor concentrations as low as 2 micrograms/l (10(-10) M) and recovery of varying amounts of bronchial inhibitor added to bronchial liquids is greater than 90%. This enzyme immunoassay appears to be a convenient way to quantify bronchial inhibitor in biological fluids such as serum, sputum, or bronchoalveolar lavage fluid.     

CAS agar diffusion assay for the measurement of siderophores in biological fluids

We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.     

A chemiluminescence automatic analyser for the measurement of biological compounds

A compact automated analyser which could analyse constituents in biological fluids with a small sample volume and in a short time has been developed. The instrument was composed of a flow injection analysis system equipped with chemiluminometric detection and an immobilized enzyme column reactor used in combination. Chemiluminescence has high sensitivity, and its reaction proceeds very quickly. Furthermore, an immobilized enzyme column reactor can produce a sufficient amount of hydrogen peroxide from compounds in serum in a short time. When enzymes are used as reagents for the analysis of substances in blood or blood serum, the final signals emitted by different enzyme reactions are usually not only hydrogen peroxide but also ammonia, NAD(P)H and so on. However, the practical chemiluminescence method for ammonia and NAD(P)H has not been established. We have discovered a new practical method for ammonia and NAD(P)H using an enzyme column reactor consisting of both immobilized L -glutamate dehydrogenase and L -glutamate oxidase. The determinations of glucose and uric acid in serum by chemiluminometry after production of hydrogen peroxide by the respective oxidases are presented. A newly chemiluminometric determination of ammonia, NAD(P)H and its applications to other enzymatic analyses that give ammonia and NAD(P)H as a final signal are also described.     

A chemiluminescent method for measurement of activated oxygen forms in biological fluids and homogenates

A chemiluminescent method for measuring the concentration of activated oxygen species (O₂² and H₂O₂) is described. Its main features are: high sensitivity (10^?9 M H₂O₂), its applicability to systems with high optical absorbance in the visible spectral region, a wide linear dynamic range, and the possibility for recording the kinetics of the processes, in which activated oxygen species are involved.     

A simple spectrophotometric method for the measurement of ribonuclease activity in biological fluids

We have developed a rapid and sensitive method for detecting ribonuclease (RNAase). The method makes use of a RNa-Pyronine Y complex which has a different absorption spectrium from that of Pyronine Y alone. When the RNA is hydrolyzed by RNAase, the spectrum of the complex changes to that of unbound Pyronine Y. The resultant decrease in absorbance at 572 nm is linear for final RNAase concentrations ranging from 2 to 45 ng/ml. Optimal assay conditions were 11.5 μg/ml Pyronine Y, 0.56 mg/ml RNA, 80 μmol/ml Tris-HCl buffer, pH 7.8 and 2–45 ng/ml RNAase. The effect of complex concentration, PH, molarity and temperature upon the rate of the reaction were determined.The assay is applicable to crude cell-free extracts.     

Xanthine analysis in biological fluids by capillary electrophoresis

Xanthine, a precursor of uric acid, is measured here in serum, urine, and cerebrospinal fluids by capillary electrophoresis (CE) after deproteinization with acetonitrile. The migration time is about 7.5 min with a minimum detection limit of 0.4 mg/l. Different purines and pyrimidines did not interfere with the determination. The method demonstrates the suitability of the CE for determination of small molecules present in a complex matrix at levels of ca. 1 mg/l. It also demonstrates that acetonitrile deproteinization is a simple and effective method for preparing samples for CE, allowing a large volume to be introduced into the capillary.     

Corticosteroid analysis in biological fluids by high-performance liquid chromatography

A sensitive, specific, and reproducible high-performance liquid chromatographic assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples are extracted with methylene chloride, washed with sodium hydroxide and then water, and chromatographed on a microparticulate silica gel column with UV detection at 254 nm. Sensitivity was greater than 15 ng for all four steroids. Specificity was supported by use of dual wavelength UV detection and/or radioimmunoassay. The assay has been applied in pharmacokinetic studies and a typical plasma concentration—time profile for the three steroids is presented for one subject who received 50 mg of prednisone.     

Determination of fosfomycin in biological fluids by capillary electrophoresis

A capillary electrophoresis method was developed for the determination of the antibiotic fosfomycin in serum, cerebrospinal fluid and aqueous humor. The technique uses indirect UV detection and the working buffer includes an organic cation to improve fosfomycin mobility. The electrophoretic time of migration is less than 7 min in both fluids. The limit of quantification is 2.5 and 1 μg/ml in serum and aqueous fluids, respectively (signal-to-noise RATIO = 3). The method was validated in serum and water over the concentration range 2.5–200 μg/ml. The calibration graph for serum was linear with a correlation coefficient r = 0.999. At a fosfomycin concentration of 2.5 μg/ml in serum, the intra- and inter-day precisions (coefficients of variation) were 5 and 5.2%, respectively. The mean recovery in serum was 94.5% (S.D. = 2.4%).     

CA 125 in biological fluids

CA 125 is not a specific tumor marker, and is synthesized by normal and malignant cells of different origin (mainly in tissues derived from the müllerian epithelia) in a similar proportion. Abnormal CA 125 levels may be found in fluids of different origin (ascites, pleura, pericardium, amniotic fluid, cyst fluid, bronchoalveolar fluid, etc.) and in serum from patients with these fluids. Differences in serum CA 125 found in malignant or benign diseases may be related to the number of cells that synthesize the marker, and are highly dependent on the access to serum, where the marker is normally determined. Moreover, CA 125 is a very good tumor marker in ovarian and lung cancer. The sensitivity of CA 125 in ovarian cancer is related to stage (40-95%), histological type (lower levels in mucinous adenocarcinoma), and the marker is useful in the early detection of recurrence (sensitivity 80%) and in therapy monitoring. It's sensitivity in lung cancer is lower than in ovarian cancer, 39% in locoregional malignancies and 69% in metastatic disease, but clearly related to stage and histology (mainly in adenocarcinomas and large cell lung cancer) and it is useful in prognosis and disease monitoring.     

Biomarker discovery in biological fluids

Discovery of novel protein biomarkers is essential for successful drug discovery and development. These novel protein biomarkers may aid accelerated drug efficacy, response, or toxicity decision making based on their enhanced sensitivity and/or specificity. These biomarkers, if necessary, could eventually be converted into novel diagnostic marker assays. Proteomic platforms developed over the past few years have given us the ability to rapidly identify novel protein biomarkers in various biological matrices from cell cultures (lysates, supernatants) to human clinical samples (serum, plasma, and urine). In this article, we delineate an approach to biomarker discovery. This approach is divided into three steps, (i) identification of markers, (ii) prioritization of identified markers, and (iii) preliminary validation (qualification) of prioritized markers. Using drug-induced idiosyncratic hepatotoxicity as a case study, the article elaborates methods and techniques utilized during the three steps of biomarker discovery process. The first step involves identification of markers using multi-dimensional protein identification technology. The second step involves prioritization of a subset of marker candidates based on several criteria such as availability of reagent set for assay development and literature association to disease biology. The last step of biomarker discovery involves development of preliminary assays to confirm the bio-analytical measurements from the first step, as well as qualify the marker(s) in pre-clinical models, to initiate future marker validation and development.     

Determination of clofibrate in biological fluids by thin-layer and gas-liquid chromatography

A specific and sensitive method is described for the detection of clofibrate in biological fluids. The drug is separated from associated fatty acids by thin-layer chromatography and the methyl ester is quantified by gas-liquid chromatography. Recovery is excellent, and any small losses are corrected with an internal recovery standard. Although more time-consuming than other available techniques, the method offers advantages for accurate studies of clofibrate metabolism.