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In this study, nanofibrous biohybrid scaffolds were developed by electrospinning using poly(ε-caprolactone) (PCL) and decellularized bovine tissue derived extracellular matrix (ECM). At the first part of the study, bovine ECM was decellularized by treatment with detergent for 24h and then combined with PCL. Following the evaluation of the decellularization efficiency via spectrophotometric DNA content analysis, the composite scaffolds were characterized by using SEM and FT-IR spectroscopy. Moreover, to assess the biocompatibility of the scaffolds an in vitro cell culture based cytotoxicity test was performed. The results indicated that, DNA content of the bovine tissue was reduced by ~80% compared to the native tissue after decellularization. While FT-IR results indicated the presence of ECM in the composite scaffolds, SEM findings showed that the porous nanofibrous structure of the scaffold changed depending on the incorporated ECM amount. Cell culture based studies also revealed that, the scaffolds containing different amounts of ECM did not have any toxic effect on cell viability during 48 hours of culture period.

OBJETIVO: Comparar la capacidad de disolución de tejido orgánico de dos soluciones (NaOCl y HOCl), sometiéndolas a diferentes protocolos de tiempo y temperatura MATERIALES y MÉTODOS: Para el presente estudio se obtuvieron 120 muestras de tejido vacuno ex vivo, el cual fue dividido en 10 grupos de 12 muestras cada uno. Se utilizaron 4 soluciones de NaOCl con concentraciones de: 5.25%, 2.5%, 1% y 0.5% respectivamente y una solución de HOCl al 5%. Las muestras fueron observadas a distintos intervalos de tiempo: 1 minuto, 5 minutos, 15 minutos, 30 minutos, 45 minutos y 60 minutos. Así los datos recogidos de las observaciones se basaron en cambios macroscópicos presentes en la superficie de cada muestra y posteriormentefueronanalizadosestadísticamenteendiferentespruebas. RESULTADOS:Los datos fueron analizados con el programa informático Statistica 7.0. El análisis 1 comparó, en los distintos intervalos de tiempo, la capacidad de disolución de tejido orgánico entre las distintas concentraciones del NaOCl y el HOCl, a los 5 minutos y a los 15 minutos, no arrojaron diferencias significativas mientras que 30 a 60 minutos, sí permitieron observar diferencias significativas. En la segunda parte del análisis estadístico se comparó, en los distintos intervalos de tiempo, la valoración entre las dos temperaturas probadas 21oC y37 oC, los resultados obtenidos para los tiempos 1 minuto, 45 minutos y 60 minutos, no permiten observar diferencias significativas. Para los intervalos de 5 minutos, 15 minutos y 30 minutos, sí se observaron diferencias, siempre a favor de las muestras tratadas a 37oC.

Background In medical training and research fresh human tissue is often replaced by preserved human or fresh animal tissue, due to availability and ethical reasons. Newer preservation approaches, such as the Thiel method, promise more realistic mechanical properties than conventional formaldehyde fixation. Concerning animal substitute material, porcine and bovine tissue is often chosen, as it is easily obtainable and certain similarity to human tissue is assumed. However, it has not been thoroughly investigated how Thiel preservation changes non-linear and viscoelastic behaviour of soft organ tissues. Furthermore, differences in these properties between animal tissue and human tissue have not been previously corroborated. Methods We conducted ramp and relaxation tensile tests on fresh human and Thiel preserved hepatic tissue, extracting strain-specific elastic moduli, and viscoelastic properties. The results for fresh human liver were then compared to corresponding results for Thiel preserved liver, as well as previously published results for porcine and bovine liver. Results Our results showed that Thiel preservation seems to be associated with increased stiffness as well as decreased viscoelastic damping behaviour. Porcine liver was stiffer than human liver with similar viscoelastic properties. Bovine liver exhibited similar stiffness as human liver, however lower viscoelastic damping. Conclusions The differences between human and animal liver tissue, concerning their mechanical properties, can be explained by their characteristic histology. Changes in mechanical properties due to Thiel preservation might stem from altered protein cross-linking and dehydration. The results illustrate that appropriate materials for medical training systems must be selected based on which mechanical properties are relevant for the respective application.

The content of total iron and total bioaccessible iron were determined in beef through visible spectrophotometry by the method of 1,10-Phenanthroline. The samples were cooked, dehydrated and prepared as jerked beef. They were submitted to a simulation of gastrointestinal digestion for the evaluation of bioaccessibility. Among the samples analyzed, the heart and the liver had the highest levels of total iron and total bioaccessible iron. After cooking the samples, there was an increased bioaccessibility of iron. The cooked liver and heart contributed respectively with 28% and 20% in terms of bioaccessibility. The insertion of bioaccessibility in food composition tables is important, as it comes to nutritional information closer to reality. Furthermore it was performed the iron speciation.

The U.S. Department of Agriculture, Food Safety Inspection Service regulatory method for monensin, Chemistry Laboratory Guidebook CLG-MON, is a semiquantitative bioautographic method adopted in 1991. Official Method of AnalysisSM (OMA) 2011.24, a modern quantitative and confirmatory LC-tandem MS method, uses no chlorinated solvents and has several advantages, including ease of use, ready availability of reagents and materials, shorter run-time, and higher throughput than CLG-MON. Therefore, a bridging study was conducted to support the replacement of method CLG-MON with OMA 2011.24 for regulatory use. Using fortified bovine tissue samples, CLG-MON yielded accuracies of 80–120% in 44 of the 56 samples tested (one sample had no result, six samples had accuracies of >120%, and five samples had accuracies of 40–160%), but the semiquantitative nature of CLG-MON prevented assessment of precision, whereas OMA 2011.24 had accuracies of 88–110% and RSDr of 0.00–15.6%. Incurred residue results corroborated these results, demonstrating improved accuracy (83.3–114%) and good precision (RSDr of 2.6–20.5%) for OMA 2011.24 compared with CLG-MON (accuracy generally within 80–150%, with exceptions). Furthermore, χ2 analysis revealed no statistically significant difference between the two methods. Thus, the microbiological activity of monensin correlated with the determination of monensin A in bovine tissues, and OMA 2011.24 provided improved accuracy and precision over CLG-MON.

Within the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantitè de Matiére (CCQM), key comparison CCQM-K141 and associated pilot study CCQM-P178 were coordinated by the National Research Council Canada (NRC). This comparison was a Track A key comparison that formed part of the OAWG 10-year strategic plan. The comparison demonstrated capabilities for measuring high-polarity analytes in a high-fat and high-protein matrix. The measurand chosen as the model system was enrofloxacin and sulfadiazine in bovine tissue. Thirteen National Metrology Institutes or Designated Institutes participated in the CCQM-K141, while two National Metrology Institutes participated in CCQM-P178. The bovine muscle tissue study material was derived from a single live animal that was administered with chemical based pharmaceutical agents prior to processing. Therefore, the study material was naturally incurred, providing a true test of extraction procedures relative to more commonly encountered spiked materials. NRC confirmed excellent homogeneity and stability of the material prior to shipping. Three 10 g bottles of freeze dried powdered muscle tissue were supplied. NRC also provided isotopically labelled solutions of the two measurands, 13C6-sulfadiazine and enrofloxacin-d5 (HI Salt), to those interested in using isotope dilution mass spectrometry (IDMS) methodologies. Procurement and purity assignment with appropriate metrological traceability of native calibrants were the responsibility of individual participants. The study required extraction, clean-up, analytical separation, and selective detection of the analytes. The level of agreement was reasonable given the measurands and matrix were new for most laboratories. The KCRV values and their uncertainties at the 95% confidence level of 57.81 ± 2.57 μg/kg for enrofloxacin and 2285 ± 68 μg/kg for sulfadiazine were calculated using the DSL means. While one participant's value was voluntarily excluded from the KCRV calculations for enrofloxacin, all other participants demonstrated equivalence for both measurands. Significant effort was undertaken post-study to identify the major sources of variability between results. In particular, the various extraction conditions used by participants were investigated thoroughly. While there appeared to be a correlation between highly acidic conditions and higher recovery, this was not definitive and could not be confirmed. The form of standards employed (i.e. free base vs salts) and potential differential solubility between forms was also a suspected source of variability. Biases could also have been introduced with the choice of solvents used for standard preparation, with some solvents better able to minimize adsorption of the analytes to glass surfaces. KEY WORDS FOR SEARCH enrofloxacin, sulfadiazine, bovine tissue, incurred, key comparison Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

The current scenario of international beef trading has increased the pressure for better and faster diagnosis of bovine tuberculosis. Although traditional culture remains the gold standard method to confirm Mycobacterium bovis infection, it is exceedingly time consuming, and demands viable mycobacteria. Molecular methods overcome the flaws of the bacteriological methods with faster detection and identification. However, mycobacterial features like a complex cell wall and pathogen–host interaction make the molecular detection a challenge. Three protocols for DNA extraction (A, B and C) from bovine tissues were tested to verify the most suitable technique for routine diagnostic assessment of their specificity and sensitivity. Thirty culture-positive and thirty culture-negative granulomatous lesions were included in the trial. From each sample, three tissue suspensions at different dilutions (10-1, 10-2 and 10-3) were prepared and submitted to DNA extraction. PCR procedures targeting IS6110 were performed, employing two volumes of DNA: 5 µL of all three dilutions, and 2.5 µL of the 10-1 dilution. Protocol A was able to detect members of the M. tuberculosis complex in most samples. The sensitivity of the test decreased with increase in tissue-suspension dilution. Although Protocol A presented the highest sensitivity followed by C and B, it showed the lowest specificity, which can be due to a failure in primary isolation caused by the lack of viable organisms or incubation time. Regardless classical bacteriological methods are still recommended by OIE, after evaluating the sensitivity of DNA extraction protocols and PCR procedures, we conclude that the best strategy for M. bovis detection is to follow Protocol A on concentrated tissue suspensions.

PURPOSE Currently, there is no accepted standard for measuring breast density. Dual energy mammography, which has demonstrated accurate measurement in phantoms, has been proposed as one possible method. To examine the use of chemical analysis as a possible means to validate breast density measurements from dual energy mammography, a bovine tissue model was investigated. Known quantities of lean and adipose tissue were compared with composition values measured from dual energy images and chemical analysis. METHODS Theoretical simulations were performed to assess the impact variations in breast composition would have on measurement of breast density from a single calibration. Fourteen ex-vivo tissue samples composed of varying amounts of pure lean tissue and pure adipose tissue (lean percentage) from 0 to 100%, in increments of 10%, were imaged using dual energy mammography. This was followed by chemical analysis based on desiccation, trituration, and fat extraction with petroleum ether to determine water, lipid, and protein content. The volumetric lean percentage (VLP) as measured from images (VLP(I)) and as derived from chemical analysis data (VLP(CA)) were compared with the VLP calculated from measurements of sample mass with a scale (VLP(M)). Finally, data from the bovine tissue model in this study were compared to compositional data from a previous report of human tissue composition. RESULTS The results from simulation suggest a substantial impact on measuring breast density is likely due to changes in anatomical breast composition. VLP(I) was related to the VLP(M) by VLP(I) = 1.53 VLP(M) + 10.0 (r2 > 0.99). VLP(CA) was related to VLP(M) by VLP(CA) = 0.76 VLP(M) + 22.8 (r2 > 0.99). VLP(I) was related to VLP(CA) by VLP(I) = 2.00 VLP(CA) - 35.6 (r2 > 0.99). Bovine adipose tissue was shown to be very similar to human adipose tissue in terms of water, lipid, and protein content with RMS differences of 1.2%. Bovine lean tissue was shown to be very similar to human skeletal muscle tissue and somewhat similar to human mammary gland tissue with RMS differences of 0.4 and 22.2%, respectively. CONCLUSIONS The results of this study show strong linear relationships between volumetric lean percentage measurements using dual energy mammography, chemical analysis and the actual mass. Determining the existence of a relationship between VLP(I) and VLP(CA) was necessary before comparing density results from the dual energy technique to composition data from chemical analysis for samples of unknown composition.

Tooth-surface pH is lowered, during drinking, to a value close to the pH of the drink itself. After the drink is swallowed, the pH rises to baseline values but this process can take several minutes. Few techniques can quantify enamel erosion at timescales representative of single drinks. The objective of this study was to compare human and bovine erosion over acid-exposure times of 2 s to 1 h. Human and bovine enamel softening was compared in vitro using nanoindentation (2-60 s of exposure to acid) and tissue loss was compared using optical profilometry (1-60 min of exposure to acid). Nanoindentation revealed statistically significant softening after 2 s (human) and 5 s (bovine); there were no significant differences in hardness reduction for the two tissues at any time-point. Profilometry demonstrated statistically significant tissue loss after 20 min (human) and 10 min (bovine); bovine tissue loss progressed 30% faster than human tissue loss. These results support the use of bovine enamel as a substitute for human enamel in erosion studies, with the understanding that for moderate exposure times bovine enamel erodes 30% faster than human enamel. Nanoindentation can be used to detect enamel dissolution at timescales comparable to the oral dwell-time of a single 'mouthful' of a beverage.

1. Masked long-chain alpha-hydroxyaldehydes were trapped in all subcellular fractions of bovine liver by application of pentafluorbenzyloxime derivatization [van Kuijk, Thomas, Stephens and Dratz (1986) Biochem. Biophys. Res. Commun. 139, 144-149] and quantified via GLC/MS using characteristic ion traces. 2. The chain-length profile of long-chain 2-hydroxyalkanales clearly indicates their relationship to plasmalogens as precursor molecules. 3. The previously postulated existence of alpha-acyloxyplasmalogens as precursor molecules of masked long-chain alpha-hydroxyaldehydes in bovine tissue lipids [Lutz and Spiteller (1991) Liebigs Ann. Chem. 1991, 563-567] was excluded. 4. The constant oxidation rate of plasmalogens in all subcellular fractions provides conclusive evidence for a non-enzymic plasmalogen epoxidation process (probably via hydroperoxy radicals). 5. The high reactivity of alpha-hydroxyaldehydes sheds some doubt on the postulation that plasmalogens protect mammalian cells against oxidative stress as postulated previously [Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11590-11596; Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11597-11606].

In der vorliegenden Arbeit wird eine Methode zur exakten Quantifizierung spezischer RNA, die kompetitive und damit quantitative Reverse-Transkriptase-Polymerase-Kettenreaktion (cRT-PCR) exemplarisch beschrieben. Im Vergleich mit konventionellen Verfahren hat diese Methode den Vorteil, das bereits geringe Mengen an Untersuchungsmaterial zur Analyse ausreichen. Die spezifischen mRNAs fur 2 Schlusselproteine des zellularen Cholesterinstoffwechsels, den Low-density-Lipoprotein(LDL)-Rezeptor und die 3-hydroxy-3-methylglutaryl-Coenzym A(HMG-CoA)-Reduktase wurde in verschiedenen bovinen Geweben gemessen. Dabei zeigte sich eine besonders hohe Expression beider mRNAs in den steroidhormonproduzierenden Geweben, i. e. Corpus luteum des Ovars und der Nebenniere, eine mittlere Expression in der Leber und eine niedrige bzw. nicht mesbare Expression im Dickdarm. Die hier am Beispiel des LDL-Rezeptors und der HMG-CoA-Reduktase exemplarisch vorgestellte Methode der quantitativen RT-PCR last sich nicht nur in der Routinediagnostik, sondern auch auf eine Vielzahl von wissenschaftlichen Fragestellungen auf dem Gebiet der Stoffwechsel-, Entzundungs- und Tumorpathologie anwenden.

Danofloxacin (DANO) residue in bovine muscle was screened at 200 ng/g by terbium-sensitized luminescence (TSL) directly measured on 10 × 6 mm C18 sorbent strips. The analyte was first adsorbed on sorbent surface by immersion in defatted homogenates. After reagent application and desiccation, TSL was directly measured on sorbent surfaces at λ(ex) = 273 nm and λ(em) = 546 nm. The luminescence intensity was linearly dependent on DANO concentration in the 0-1000 ng/g range (R(2) = 0.9967). A threshold was established at x(200) - 3σ(200), where x(200) and σ(200) are the mean and standard deviation, respectively, of the DANO signals at 200 ng/g. Among 48 blind samples randomly fortified at 0-1000 ng/g, 45 were screened correctly and 3 negative samples were presumed positive. This simple screening protocol has the potential to significantly reduce sample numbers and hence improve sample throughput and save assay costs.

STR-based individualisation of biological deposits on bullets after perforation of tissue, can identify the person injured or killed by a particular bullet and comparison with the firearms used can identify the weapon and thus possibly the person who did the shooting. In this study, the effect of subsequent impacts on intermediate targets such as loss of cells was investigated by amplification of mitochondrial (mt) DNA. Bovine tissue was perforated and the 9 mm Luger FMJ bullets were recovered from the bullet collector. The mt cytochrome-b (cyt-b) gene could be amplified by the polymerase chain reaction (PCR) from 14 out of 15 bullets. Examination with a scanning electron microscope (SEM) and an energy-dispersive X-ray spectrometer (EDS) demonstrated the presence of minute dried tissue deposits on all bullets (n = 10) but was not able to establish preferential locations. In a series of 25 gunshots, various intermediate targets (glass, wood, car metal, gypsum board, asphalt) were perforated/ impacted following perforation of tissue and the cyt-b gene could be typed from all bullets. It is concluded that subsequent impacts on intermediate targets do not eliminate enough biological deposits to render DNA analysis impossible and that the amplification of mtDNA is a useful additional method.

A variant of laminin has previously been isolated from murine heart and shown to be distinct from laminin purified from a traditional source, the murine Engelbreth-Holm-Swarm (EHS) tumor (Paulsson, M., and Saladin, K. (1989) J. Biol. Chem. 264, 18726-18732). It contains a novel polypeptide chain designated as 300 kDa, which is not found in laminin from the EHS tumor. In the present study, heart laminin was purified from bovine tissue and shown to be structurally and immunochemically closely related to the murine protein. Further, heart laminins were compared with merosin, a laminin-like protein isolated from human placenta (Ehrig, K., Leivo, I., Argraves, W. S., Ruoslahti, E., and Engvall, E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3264-3268). The 300-kDa chain of bovine heart laminin cross-reacted with the heavy chain of merosin, showing that these polypeptides are closely related, albeit from different species. Heart laminin is more resistant to proteolysis than laminin derived from the EHS tumor. A large fragment could be prepared by digestion with thermolysin, which consisted of an almost intact long arm structure and variably long, residual short arm structures. Analysis of its structure shows that the 300-kDa heavy chain is disulfide-bonded to the B1 and B2 chains in the center of the laminin cross and forms the long arm together with these chains. It thereby replaces the A chain, well known from tumor sources, in the laminin structure.

Influence of pH, salt, phosphate, cooking temperature, muscle variation, carcass sex and maturity on lactate dehydrogenase (LDH, E.C. 1.1.1.27) activity in bovine tissue slurries was investigated. LDH activity declined sharply as temperature neared 63°C at pH 5.6 and 6.4, but no activity was detected at pH 4.8. As salt and phosphate concentrations increased, LDH activity decreased. LDH activity varied greatly from muscle to muscle. Carcasses did not differ in LDH activity by sex, but showed a decrease in activity as maturity increased. LDH has potential as a determinant of the minimum heating endpoint in precooked beef and a rapid analytical test could be developed.

This study presents Pd determinations in bovine tissue samples containing palladium prepared in the laboratory, and CCQM-P63 automotive catalyst materials of the Proficiency Test, using instrumental thermal and epithermal neutron activation analysis and energy dispersive X-ray fluorescence techniques. Solvent extraction and solid phase extraction procedures were also applied to separate Pd from interfering elements before the irradiation in the nuclear reactor. The results obtained by different techniques were compared against each other to examine sensitivity, precision and accuracy.

Background: Body-composition assessment reflects infant growth and nutritional status but is limited by practical considerations, accuracy, and safety. Objective: This study evaluated the precision and accuracy of a new air-displacement plethysmography (ADP) system for pediatric body-composition assessment. Design: We used 24 phantoms constructed from bovine lean muscle and fat. The phantoms varied in mass (1.3894-9.9516 kg) and percentage fat (%Fat; 2.08-34.40%), thereby representing infants between birth and 6 mo of age. Estimates of %Fat obtained with chemical analysis (CA), hydrostatic weighing, and ADP were compared. Results: There was no significant difference between %Fat measured with ADP (%Fat ADP ) and %Fat measured with CA (%Fat CA ); the mean values were 18.55% and 18.59%, respectively. SDs for %Fat ADP and %Fat CA were not significantly different (0.70% and 0.73%, respectively). %Fat measurements obtained with ADP, CA, and hydrostatic weighing were highly correlated (r > 0.99, P < 0.0001). The regression equation (%Fat CA = 0.996%Fat ADP + 0.119; SEE = 0.600; adjusted R 2 = 0.997; P < 0.0001) did not differ significantly from the line of identity (%Fat CA = %Fat ADP ). There was high agreement between individual measurements of %Fat ADP and %Fat CA , as shown by the narrow 95% limits of agreements between methods (-1.22% to 1.13%), and there was no systematic bias in individual differences across the phantom mass and %Fat ranges. Conclusion: ADP provides a highly precise and accurate estimate of %Fat in bovine tissue phantoms in the pediatric ranges of body weight and body fatness.

The use of infrared light (IRL) has been proposed as a replacement of the Nd:YAG laser in several contact surgeries, for example, hemorrhoids. Although the photo coagulant effect can indeed be obtained with the IRL, we found that the thermal degradation (loss of one-third of the temperature gain) is profoundly altered when using IRL, and this could have important negative side effects in the normal surrounding tissues.

A simple and elegant method using disposable silica gel columns has been developed to separate the phospholipid fraction in beef muscles from other fatty acid containing lipids. By this method, 10 samples can be cleaned up simultaneously in less than 2 h. It does not require complicated equipment. The separation of the phospholipid fraction is necessary in order to characterize it by chemical means, for instance to determine the fatty acid pattern. Recovery and reproducibility were proved with a model meat system. Purity was proved with a model system and biological samples.

Comparison of high performance liquid chromatograms (HPLC) with capillary electrophoresis shows the latter to be superior in many cases owing to rapid separation, high resolution and high sensitivity. This is demonstrated with two examples (i) the isolation of natural peptides from bovine tissue and (ii) characterization of a synthetic peptide mixture with the natural sequence (fragment) of the human immunodeficiency virus (HIV) transmembrane glycoprotein gp 41 env.

The presence of vitamin K-dependent carboxylase was investigated in the microsomal fraction of 20 different types of bovine tissue. Except for muscle, veins, lymphocytes and bone membrane, carboxylase was found in all these preparations, albeit in varying amounts. No differences could be detected between these carboxylating systems with respect to their affinity for vitamin K and warfarin. Most of the endogenous substrates had some affinity towards antiprothrombin or antifactor X.

The objective of this study was to evaluate near infrared reflectance spectroscopy (NIRS) as an accurate and inexpensive alternative to conventional chemical analyses of nonconsumer bovine tissue. Udder, plate and visceral samples were collected from mature, Charolais-Angus and Hereford-Angus crossbred beef cows at slaughter, ground and analyzed for concentrations of lipid, protein and dry matter using standard AOAC chemical procedures. Samples were analyzed using NIRS. The collection of samples was randomly split into separate calibration and validation sets. Nine calibration equations representing each constituent and tissue combination were developed, using first- or second-order derivative mathematical transformations, and those calibration equations were validated. Correlation coefficients of calibration (R) and validation (r) ranged from .95 to .98 and from .87 to .97, respectively, for all analyses except plate dry matter (r = .77). Standard errors of calibration and prediction ranged from 1.89 to 5.81%. Results from this study are interpreted to indicate that bovine udder, plate and visceral tissue composition can be accurately, quickly and efficiently predicted using NIRS technology.

A method is described for the isolation and density gradient enrichment of mammary epithelial fragments from pregnant, nonlactating bovine tissue. Immunocytochemical analysis prior to and following culture revealed specific staining with antibodies to keratin, indicating that these cells are epithelial in nature. Fragments enriched for epithelium could be stored in liquid nitrogen for extended periods prior to culture. When cast within a three-dimensional matrix of collagen gel, the mammary fragments grew as branching, duct-like structures and displayed a 4-fold increase in cell number during 10 to 12 d of culture.

Seven laboratories collaboratively studied a method for the assay of melengestrol acetate at the 0, 10, and 20 ppb levels in bovine fat, liver, muscle, and kidney. The study included fortification of tissue by each laboratory and analysis of fat samples taken from treated heifers which had endogenous levels of 0, 10, and 20 ppb melengestrol acetate. The multistep cleanup procedure used included extraction, solvent partition, column chromatography, and electron capture gas-liquid chromatographic determination. Results of the study for muscle, liver, kidney, and fat showed that the method gave satisfactory recoveries and accuracy. In fat, the most critical tissue, recovery was >93%. A statistical comparison of the results reported for fat tissue from treated heifers demonstrated that 5 of the 7 laboratories obtained similar results. The results produced by the method can be expected to be repeatable within and among laboratories. On the basis of the collaborative results the method has been adopted as official first action.

Radioimmunoassay (RIA) is valuable for sensitive and specific routine measurements of hormones in body fluids. The method is also applicable to determine tissue levels of hormones which, according to tracer studies and depending on the compound, are scattered in the lower ng and pg/g range. For practical assay application it must be decided whether the parent compound or a major metabolite should be measured. Both toxicological and practical regulatory aspects have to be considered, but in any case knowledge of the metabolic profile of the compounds is necessary, especially since the specificity of the RIA group analysis is limited. When applying extraction and purification steps, differences between compounds and between tissues have to be considered; generally more difficulties are encountered than with plasma. Validation of the method should first demonstrate no unspccific interference with the antigen-antibody reaction; then the other reliability criteria have to be established. So far RIAs for the quantitation of estrogens, testosterone, progesterone, and trienbolone—a synthetic androgen—in various bovine tissue samples have been developed. The lower limit of sensitivity ranges between 10 and 70 pg/g. Quantitation of physiological levels of testosterone and progesterone is possible, but there are still some problems with estrogens.

SummaryAn abnormal blood coagulation factor IX has been isolated from the blood of a hemophilia B patient with a variant of the disease (hemophilia Bm) characterized by a normal concentration of factor IX antigen, negligible factor IX coagulant activity, and a prolonged prothrombin time with bovine tissue factor. The isolated protein (factor IXBm) had the same apparent molecular weight as normal factor IX (55,000) and the same mobility on two dimensional immunoelectrophoresis as normal factor IX. Factor IXBm underwent limited proteolysis induced by activated factor XI, in the presence of Ca2+ ions, or induced by the reaction product of tissue factor, factor VII and Ca2+ ions. A timecourse study showed that activated factor XI cleaved factor IXBm and factor IX at similar rates. However, in contrast to normal factor IX, the limited protelysis of factor IXBm did not generate procoagulant activity.In kinetic experiments purified factor IXBm behaved like a competitive inhibitor (Ki of 0.017 μM) of the activation of factor X by bovine tissue factor and factor VII. Normal factor IX was also found to inhibit the reaction but required a four-fold higher concentration to achieve the same inhibitory effects as factor IXBm.

Samples of the pork, veal and chicken produce imported into Italy from other EEC countries were taken and analysed to ascertain saturated and unsaturated hydrocarbon levels. Gas chromatography—mass spectrometry analysis showed overall n -alkane levels to be in the range of 0·3 to 10·5 ppm. N -alkanes ranged from C 12 to C 33 . Phytenes were only found in bovine tissue. No alkenes were present in any of the samples analysed.

1. 1. The concentration of the branched-chain hydrocarbons, pristane and phytane, was determined in a number of human and animal tissues by quantitative thin-layer and gas chromatography. 2. 2. In human serum and liver, pristane was found at concentrations of 2–9 μg/g, and of 52 μg/g in a single sample of human skin. The amounts of phytane were significantly lower and represented 1–35% of those in pristane in the various tissue samples analyzed. 3. 3. Rat liver and a number of bovine tissue samples contained similar concentrations of pristane and phytane to that found in human organs. 4. 4. Pristane was found in livers from a number of shark species at a concentration of 2–5 μg/g and, in contrast to the mammalian tissues, phytane was present at similar concentrations to those of pristane. 5. 5. The possible sources of these and other hydrocarbons found in animals are briefly discussed.

11β,17α, 21-Trihydroxy-4-pregnene-3, 20-dione-4-14C when incubated with an acetone powder of bovine adrenal cortex microsomes in the presence of NADH at a pH of 5.8 was converted to 3β,11β,17α, 21-tetrahydroxy-5-pregnen-20-one-14C. The 3β-hydroxysteroid dehydrogenase-isomerase reactions in bovine tissue are therefore reversible, and the biochemical formation of 33,11β,17α, 21-tetrahydroxy-5-pregnen-20-one is definitively established.

The whole figure of the present data will be given as, a whole once in the tabular form, Tab. VI, and again briefly in the following paragraphs. The medulla of cattle, horses and dogs involves the adrenaline, determinable by the chemical methods, as the Folin, the Suto, the Stuber and the Shaw, in about 1% of tissue and that by the rabbit intestine seg-ment method in about 1.5%. But, exactly examined, the values obtained by these chemical methods not only do not check with each other, but there are also definite differences not negligible. And they are different accord-ing to the animal species, as readily seen in the summary table. On the whole, while the Suto method yields commonly the smallest .amount of adrenaline, or always a smaller amount than the Folin, the: Stuber and the rabbit intestine segment, the Shaw method gives to the bovine and horse suprarenal medullary tissue a similar value to the Suto, but to the dog medullarly tissue and rabbit suprarenal a definitely smaller -one than the Suto method. Notwithstanding that the Suto method and the Stuber are similarly the reaction with mercuric chloride, and they give quite the same amount of adrenaline to the dog's suprarenal medulla and to the rabbit suprarenals, they give on the other hand dissimilar value for the bovine and horse medullary tissues, such as 11% by the Stuber and 10% by the Suto for the bovine tissue and 11.5% by the Stuber and 10.5% by the Suto for the horse tissue. Briefly recapitulated: The methods of Stuber et al and of Shaw have been further added in solving the problem how the active principles in the suprarenal medulla, which acts as adrenaline or epinephrine, both physio-logically and chemically, are to be appreciated duly. They gave us then further evidences for our conviction that it is not legitimate to assume -only an active principle such as the adrenaline or epinephrine exists solely there, but the matter is of much complex nature.

Chloramine-T and sodium borate solutions were evaluated for their effectiveness in preserving Mycobacterium bovis and controlling the growth of non-mycobacterial contaminants on tissue specimens during transport to laboratories. The number of culturable M. bovis cells in suspension was reduced by 5.1 log10 upon exposure to chloramine-T solution and by less than 1 log10 upon exposure to sodium borate solution for 7 days. Reinoculation of laboratory media (because of overgrowth by non-mycobacterial contaminants) was required for 52.6% of 190 routine bovine tissue specimens shipped refrigerated in chloramine-T solution and for 6.1% of 520 specimens shipped unrefrigerated in sodium borate solution. M. bovis was isolated from bovine tissue stored in sodium borate solution at 23 degrees C for 17 weeks and at 4 degrees C for 25 weeks. Unrefrigerated sodium borate solution has been used successfully to ship tissue specimens to our laboratory for the past 11 years.

An electron capture GLC method has been developed for the determination of melengestrol acetate (MGA) in frozen bovine tissue, which removes, through extraction, solvent partition, and chromatographic cleanup, most of the interfering materials before injection of the sample onto the column for detection. MGA is extracted from lean tissue with acetonitrile and partitioned against hexane to remove nonpolar impurities. MGA in fatty tissues is first extracted with hexane and then transferred into acetonitrile. The residue from either extract, after evaporation of the solvent, is chromatographed on an alumina column with hexane and a mixture of hexane-acetone to remove the interfering lipids. The MGA is then eluted with hexane-chloroform (1+1). The residue, after rotary evaporation, is dissolved in the appropriate volume of hexanemethanol (94+6) and injected onto a 2′ 1% OV-17 column in an all-glass system. MGA can be detected at 25 ppb (2–3 mm peak) with no interference from tissue or reagents. The observed recovery of MGA at 25 ppb in muscle, liver, fat, kidney, and bone marrow was 93 ±7%.

F.IXBm is giving a prolonged prothrombin time with bovine tissue factor (T.F.). In the present study the inhibitory effect of purified F.IXBm was tested in a F.VII assay that measures the ability of F.VII to activate F.X in the presence of bovine T.F. and Ca++, followed by the quantitation of formed F.Xa by the synthetic substrate benzoyl -L-Ile-Glu-Gly-L-Arg-p-nitroanilide. Experiments were carried out with different concentrations of F.IXBm and F.X, keeping the concentrations of F.VII and bovine T.F. constant. Lineweaver-Burk plot of the data showed that all the curves crossed the l/V axis in one point - no inhibition by F.IXBm at infinite concentratlons of F.X. Thus, F.IXBm acts as competitive in inhibitor to F.X in the activation of F.X by F.VII and bovine T.F. By plotting the 1/V against the inhibitor concentrations (F.IXBm conc), a Dixon plot, the Ki for F.IXBm was determined to be 0.017 μM.Normal F.IX was also found to be a competitive inhibitor to F.X but 4-5 times higher concentrations of F.IX were needed to give the same inhibition as F.IXBm. Utilizing human T.F. instead of bovine T.F. in this assay resulted in no significant inhibition by either F.IX of F.IXBm.

A sensitive high-pressure liquid chromatographic assay procedure is described for residues of sulfamethazine in bovine kidney, liver, muscle, and fat tissue at a sensitivity of 0.04 ppm. Studies were conducted using sulfamethazine-negative bovine tissue to ensure that the peak eluting at the indicated retention volume was sulfamethazine and that the recoveries from spiked tissues were consistent. Data are presented showing the levels of sulfamethazine in the tissue of 12 treated animals found by this technique. The method is more specific than the frequently used colorimetric methods and does not require the tedious separations involved in TLC assays.

1. 1. Immunolochemical studies are presented which support the suggestion (Ma & Fisher, 1969) that the large and small adenosine deaminases in bovine tissues are different aggregated states of the same monomeric enzyme. 2. 2. Also, it is shown that four of the bovine tissue contain principally the small enzyme, three contain the large enzyme and one contains a mixture of both.

An extraction and cleanup procedure is described for the analysis of cyclofenil in bovine muscle tissue. Flame ionization gas chromatography is used as the determinative step. An average recovery of 95% cyclofenil was obtained for tissue fortified with 0.5–30 μg cyclofenil/25g sample.

A mixture of fatty acids obtained from autolyzed saline extracts of beef testicular tissue was fractionated by crystallization of the urea complexes at 5°, −20°, and −75°C. Fractions rich in docosahexaenoic and in arachidonic acids were obtained as solid complexes. The filtrate remaining after precipitation of the solid urea complexes contained a high percentage of hexaenoic acid of shorter chain length than docosahexaenoic, probably eicosahexaenoic acid.