Sheep Plasma Research Articles

Development of a polar lipid profiling method by supercritical fluid chromatography/mass spectrometry

We established a high-throughput and high-resolution analytical method based on supercritical fluid chromatography (SFC) coupled with mass spectrometry (MS) for the simultaneous profiling of diverse polar lipids in a mixture. Trimethylsilyl (TMS) derivatization was used for the analysis of ten polar lipids: phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidic acid (LPA), lysophosphatidylinositol (LPI), sphingomyeline (SM), and sphingosine-1-phosphate (S1P). Using the developed method, the peak tailings of PA, PI, LPA, LPI, and S1P improved, and the limit of detection of PG, PI, LPA, LPI, and S1P was enhanced by 12-, 40-, 510-, 39-, and 1490-fold, respectively. Next, in the analysis of sheep plasma, 20 minor species of PI, LPC, LPE, and SM, and 7 molecular species of LPA, LPI, and S1P were additionally analyzed. The relative ratio of the molecular species in each polar lipid was also found by quantification. Finally, the simultaneous and detail profiling of ten polar lipids was successfully performed by SFC/MS applying TMS derivatization. This developed method is particularly applicable to metabolomics, especially for targeting polar lipids.

Epitope Mapping of a Monoclonal Antibody to Factor VIII That Inhibits Factor IXa:Factor VIIIa Interaction and Thrombin Activation of Factor VIII

Abstract 1177Factor VIII (FVIII) circulates in plasma as a noncovalent heterodimer consisting of a heavy chain (HC, A1-a1-A2-a2-B domains) and a light chain (LC, a3-A3-C1-C2 domains) in a noncovalent complex with von Willebrand factor (wVF). Thrombin (IIa) cleaves FVIII between the A1-a1/A2 domains at Arg372, A2-a2/B domains at Arg740 and B-a3/A3 domains at Arg1689 generating FVIIIa that consists of an A1-a1/A2-a2/A3-C1-C2 heterotrimer. FVIIIa increases the efficiency of Factor IXa (FIXa) catalyzed activation of Factor X (FX) in a Ca2+ and phospholipid (PL) dependent manner. The A3-C1-C2 segment of FVIIIa plays an important role in FIXa:FVIIIa interaction. Here, we describe a series of experiments to map the epitope of a monoclonal antibody (mAb) that is reported to inhibit FVIII clotting activity in a one stage clotting assay (Brown et al; J Lab Clin Med, 101: 793–805, 1983).The binding of mAb to FVIII, B-domain deleted FVIII and isolated LC was assessed using surface plasmon resonance. In these experiments, mAb captured on a protein A/G coupled CM5 sensor chip served as the ligand, and FVIII and its isolated fragments served as the analytes. The Kd of binding of LC (∼40 nM) was similar to FVIII and the B-domain deleted FVIII. No binding was observed for isolated A1 and A2 domains. Further, in plasma based inhibition assays, the Kd of binding of mAb to FVIII-vWF complex and to FVIII was ∼30 nM. This suggests that the mAb epitope does not significantly overlap with the vWF binding site in the acidic a3 region of LC. Western blot analysis confirmed that the mAb is specific for the LC of FVIII. Moreover, IIa-cleaved LC starting at residue 1690 gave only a weak signal and FXa-cleaved LC starting at residue 1721 did not react with the mAb in Western blots. These data suggest that the epitope for this mAb spans the IIa-cleavage site in the LC. Consistent with these observations, the A3-C1-C2 fragment but not the C1-C2 fragment expressed in COS cells reacted with the mAb. To further define a part of the epitope in the IIa-cleaved LC, twelve A3 domain deletion fragments were constructed and expressed in E. coli. Western blot analysis of these fragments restricted the partial epitope to 1690–1710 residues of the IIa-cleaved LC. In additional experiments, the mAb did not inhibit mouse, rabbit or canine plasma FVIII in a one stage clotting assay. It did however inhibit porcine plasma FVIII with ∼40 nM Kd, sheep plasma FVIII with ∼ 68 nM Kd, and bovine plasma FVIII with ∼300 nM Kd. Analysis of the sequence alignment of residues 1680 to 1710 of FVIII from each species indicated that residues 1681 to 1694 of human FVIII most likely constitute the epitope of this mAb. The dissimilarity and the charge differences in amino acids suggest that residues Asp1681, Glu1684, Asn1685, and Ser1687 on the N terminal side and Lys1693 on the C terminal side of the IIa-cleavage site Arg1689-Ser1690 may be important for this epitope.Fluorescence energy transfer (FRET) experiments indicated that the mAb inhibits FIXa interaction with the IIa-cleaved LC consisting of A3-C1-C2 domains. In these experiments, A3-C1-C2 subunit was labeled with acrylodan (fluorescence donor) and FIXa was labeled with fluorescein-Phe-Phe-Arg-chloromethylketone (fluorescence acceptor). In the presence of FIXa, the acrylodan fluorescence was quenched indicating a biomolecular complex formation. Addition of 1.2 μM mAb abolished the acrylodan fluorescence quenching suggesting inhibition of the FIXa:LC interaction. Notably, the mAb did not inhibit activation of FX by FIXa/Ca2+/PL and FXa-cleaved FVIIIa (instead of IIa-cleaved FVIIIa). This suggests that the mAb inhibits FIXa:LC interaction by a steric hindrance and not by a direct blockage of the FIXa:LC interactive sites.In summary, the mAb inhibits clotting by preventing FVIII activation by IIa. The epitope of the mAb appears to be restricted to residues 1681–1694 of FVIII. Notably, in some of the hemophilia A patients, the epitope of the inhibitory antibodies is confined to the IIa-cleavage site including the a3 acidic domain of LC. To locate the epitope for such antibodies, one of the approaches used was to construct porcine and human FVIII hybrids. Our strategy may represent a simplified approach to locate the epitope of similar antibodies in hemophilia A patients. Such antibodies may bind strongly to LC and weakly to IIa-cleaved LC. Further, these antibodies may not bind to FXa-cleaved LC or A1/A2 subunits. Disclosures:No relevant conflicts of interest to declare.

Changes in concentrations of lysergol in urine and prolactin in plasma, rectal temperature and respiration rate in sheep selected for resistance or susceptibility to ryegrass staggers and fed ergovaline

AIM: To determine the effects of feeding ryegrass seed containing ergovaline to sheep selected for resistance or susceptibility to ryegrass staggers on concentration of lysergol (a metabolite of ergovaline) in urine, prolactin in plasma, rectal temperature and respiration rate. METHODS: Two experiments were carried out using 12 Romney crossbred ewe lambs aged 9 months, comprising animals resistant (n=4), susceptible (n=4) or outcross (n=4) to ryegrass staggers. In Experiment 1, sheep were given either a single (Part A) or six (Part B) feed (s) of endophyte-infected seed containing ergovaline at 30 mg/kg dry matter (DM), at 42 μg ergovaline/kg bodyweight (BW), to simulate acute and chronic exposure to ergovaline, respectively. The concentration and excretion of lysergol in urine and concentration of prolactin in plasma were measured over 3 and 12 days, for Parts A and B respectively. In Experiment 2, after a recovery period of 7 days, the same sheep were fed with ergovaline at 67 μg/kg of BW for 7 days. Soon after the seventh feed the sheep were moved to a hothouse at 36.5°C and 60% humidity, and 3 h later their rectal temperatures and respiration rates were measured. RESULTS: The concentration of lysergol and excretion in urine increased to a peak between 6 and 9 h after exposure to ergovaline whereas the concentration of prolactin in plasma was markedly reduced. Differences in concentration and rate of excretion of lysergol in urine between animals resistant, outcross and susceptible to ryegrass staggers were not significant (p>0.1). The animals resistant to ryegrass staggers had a lower rectal temperature (p<0.05) and a faster respiration rate than the outcross and susceptible groups when exposed to high ambient temperature and high humidity. CONCLUSIONS: This study showed that excretion of lysergol in urine increased with each exposure of sheep to ergovaline. Animals genetically resistant to ryegrass staggers exhibited a lower rectal temperature and a faster respiration rate than those susceptible, demonstrating the possible cross resistance of sheep to ergovaline in a population originally selected for resistance to ryegrass staggers. Hence potential exists to select animals resistant to ryegrass staggers that are also resistant to ergovaline.

Response to Letter Regarding Article, “B-Type Natriuretic Peptide Signal Peptide Circulates in Human Blood: Evaluation as a Potential Biomarker of Cardiac Ischemia”

We thank Drs Goetze and Kavsak and their respective colleagues for their interest in our study.1 Our identification of signal peptide of B-type natriuretic peptide (BNPsp)(17-26) was achieved through specific immunoaffinity purification followed by multiple tandem mass spectrometry with the use of the same antibody that is present in the assay. Cross-reactivity data demonstrated negligible interference from many other peptides, including other human peptide signal sequences and standard medications. Rat and sheep plasma samples display no observable immunoreactivity. Analysis of the BNPsp(17-26) amino acid sequence on the National Center for Biotechnology Information protein Basic Local Alignment Search Tool reveals that only the signal peptide of human …

Simple HPLC method for determination of rosiglitazone in sheep plasma and amniotic fluid and its application in a pregnant sheep model

This paper describes the development of a sensitive high performance liquid chromatography (HPLC) method for quantification of rosiglitazone in sheep plasma and amniotic fluid. Samples were prepared by liquid–liquid extraction using tert-butyl methyl ether, and rosiglitazone was quantitated by HPLC using a C18 column and fluorescence detector with an excitation wavelength of 247 nm and emission wavelength of 367 nm. The mobile phase consisted of ammonium acetate (10 mM, pH 5.2) and acetonitrile (56.5:43.5, v/v) with a flow rate of 1 ml/min. Ketoconazole was used as the internal standard (IS). The plasma calibration curve was linear over the range of 2.5–250 ng/ml (mean r 2 = 0.9940 ± 0.0024; n = 6) with accuracy of 99.4–102.8% over the calibration range. The intra-day and inter-day coefficient of variation (%CV, percent coefficient of variation) were in the range of 0.01–8.68% in sheep plasma. Similar performance was achieved for amniotic fluid. The described method was successfully applied to quantitate rosiglitazone concentrations in the pregnant ewe and her fetus.

Development and validation of a liquid chromatography and ion spray tandem mass spectrometry method for the quantification of artesunate, artemether and their major metabolites dihydroartemisinin and dihydroartemisinin‐glucuronide in sheep plasma

Recently, promising fasciocidal activities of artesunate and artemether were described in rats and sheep. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify artesunate, artemether and their metabolites dihydroartemisinin and dihydroartemisinin-glucuronide in sheep plasma. Protein precipitation with methanol was used for sample workup. Reversed-phase high-performance liquid chromatography (HPLC) was performed using an Atlantis C18 analytical column with a mobile phase gradient system of ammonium formate and acetonitrile. The analytes were detected by MS/MS using selected reaction monitoring (SRM) with electrospray ionisation in the positive mode (transition m/z 267.4 → 163.0). The analytical range for dihydroartemisinin, dihydroartemisinin-glucuronide and artesunate was 10-1000 ng/ml and for artemether 90-3000 ng/ml with a lower limit of quantification of 10 and 90 ng/ml, respectively. Inter- and intra-day accuracy and precision deviations were < 10%. Consistent relative recoveries (60-80%) were observed over the investigated calibration range for all analytes. All analytes were stable in the autosampler for at least 30 h (6 °C) and after three freeze and thaw cycles. The validation results demonstrated that the LC-MS/MS method is precise, accurate and selective and can be used for the determination of the artemisinins in sheep plasma. The method was applied successfully to determine the pharmacokinetic parameters of artesunate and its metabolites in plasma of intramuscularly treated sheep.

Classical scrapie prions in ovine blood are associated with B lymphocytes and platelet-rich plasma

BackgroundClassical scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats characterized by cellular accumulation of abnormal isoforms of prion protein (PrPSc) in the central nervous system and the follicles of peripheral lymphoid tissues. Previous studies have shown that the whole blood and buffy coat blood fraction of scrapie infected sheep harbor prion infectivity. Although PrPSc has been detected in peripheral blood mononuclear cells (PBMCs), plasma, and more recently within a subpopulation of B lymphocytes, the infectivity status of these cells and plasma in sheep remains unknown. Therefore, the objective of this study was to determine whether circulating PBMCs, B lymphocytes and platelets from classical scrapie infected sheep harbor prion infectivity using a sheep bioassay.ResultsSerial rectal mucosal biopsy and immunohistochemistry were used to detect preclinical infection in lambs transfused with whole blood or blood cell fractions from preclinical or clinical scrapie infected sheep. PrPSc immunolabeling was detected in antemortem rectal and postmortem lymphoid tissues from recipient lambs receiving PBMCs (15/15), CD72+ B lymphocytes (3/3), CD21+ B lymphocytes (3/3) or platelet-rich plasma (2/3) fractions. As expected, whole blood (11/13) and buffy coat (5/5) recipients showed positive PrPSc labeling in lymphoid follicles. However, at 549 days post-transfusion, PrPSc was not detected in rectal or other lymphoid tissues in three sheep receiving platelet-poor plasma fraction.ConclusionsPrion infectivity was detected in circulating PBMCs, CD72+ pan B lymphocytes, the CD21+ subpopulation of B lymphocytes and platelet-rich plasma of classical scrapie infected sheep using a sheep bioassay. Combining platelets with B lymphocytes might enhance PrPSc detection levels in blood samples.

Biodynamic parameters of micellar diminazene in sheep erythrocytes and blood plasma

In this work, we used a preparation of diminazene, which belongs to the group of aromatic diamidines. This compound acts on the causative agents of blood protozoan diseases produced by both flagellated protozoa (Trypanosoma) and members of the class Piroplasmida (Babesia, Theileria, and Cytauxzoon) in various domestic and wild animals, and it is widely used in veterinary medicine. We examined the behavior of water-disperse diminazene (immobilized in Tween 80 micelles) at the cellular and organismal levels. We assessed the interaction of an aqueous and a water-disperse preparation with cells of the reticuloendothelial system. We compared the kinetic parameters of aqueous and water-disperse diminazene in sheep erythrocytes and plasma. The therapeutic properties of these two preparations were also compared. We found that the surface-active substances improved intracellular penetration of the active substance through interaction with the cell membrane. In sheep blood erythrocytes, micellar diminazene accumulated more than its aqueous analog. This form was also more effective therapeutically than the aqueous analog. Our findings demonstrate that use of micellar diminazene allows the injection dose to be reduced by 30%.

Development and Validation of a High-Performance Liquid Chromatography Method for Determination of Cefquinome Concentrations in Sheep Plasma and Its Application to Pharmacokinetic Studies

Cefquinome has a broad spectrum of antibacterial activity and was developed especially for use in animals. A simple and sensitive high-performance liquid chromatography (HPLC) method with UV-visible detection for quantification of cefquinome concentrations in sheep plasma was developed and validated. Separation of cefquinome from plasma components was achieved on a Phenomenex Gemini C(18) column (250 mm by 4.6 mm; internal diameter [i.d.], 5 μm). The mobile phase consisted of acetonitrile and 0.1% trifluoroacetic acid in water and was delivered at a rate of 0.9 ml/min. A simple and rapid sample preparation involved the addition of methanol to 200 μl of plasma to precipitate plasma proteins followed by direct injection of 50 μl of supernatant into the high-performance liquid chromatography system. The linearity range of the proposed method was 0.02 to 12 μg/ml. The intraday and interday coefficients of variation obtained from cefquinome were less than 5%, and biases ranged from -3.76% to 1.24%. Mean recovery based on low-, medium-, and high-quality control standards ranged between 92.0 and 93.9%. Plasma samples were found to be stable in various storage conditions (freeze-thaw, postpreparative, short-term, and long-term stability). The method described was found to be readily available, practicable, cheap, rapid, sensitive, precise, and accurate. It was successfully applied to the study of the pharmacokinetics of cefquinome in sheep. This method can be very useful and an alternate to performing pharmacokinetic studies in the determination of cefquinome for clinical use.

Determination of Adenosine and Inosine in Sheep Plasma Using Solid Phase Extraction Followed by Liquid Chromatography with UV Detection

A simple and sensitive liquid chromatography assay following solid phase extraction was developed for simultaneous determination of adenosine and inosine in sheep plasma. The system consisted of a Symmetry C18 column, a mobile phase composed of acetonitrile, 100 mM sodium dihydrogen phosphate and water, and ultraviolet detection at 254 nm. The method showed good sensitivity (limits of detection for adenosine and inosine were 30 and 50 ng/ml, respectively, in the plasma samples), repeatability, and linearity. The developed method was applied to sheep plasma samples from a study examining the cardio active potential of the combination of adenosine and inosine.

Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples

The widespread occurrence of heparin contaminated with oversulfated chrondroitin sulfate (OSCS) in 2008 initiated a comprehensive revision process of the Pharmacopoeial heparin monographs and stimulated research in analytical techniques for the quality control of heparin. Here, a set of 177 heparin samples from the market in 2008 as well as pure heparin sodium spiked with defined amounts of OSCS and DS were used to evaluate established and novel methods for the quality control of heparin. Besides (1)H nuclear magnetic resonance spectroscopy (NMR), the assessment included two further spectroscopic methods, i.e., attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy, three coagulation assays, i.e., activated partial thromboplastin time (aPTT) performed with both sheep and human plasma and the prothrombin time (PT), and finally two novel purity assays, each consisting of an incubation step with heparinase I followed by either a fluorescence measurement (Inc-PolyH-assay) or by a chromogenic aXa-assay (Inc-aXa-assay). NMR was shown to allow not only sensitive detection, but also quantification of OSCS by using the peak-height method and a response factor determined by calibration. Chemometric evaluation of the NMR, ATR-IR, and Raman spectra by statistical classification techniques turned out to be best with NMR spectra concerning the detection of OSCS. The validity of the aPTT, the current EP assay, could be considerably improved by replacing the sheep plasma by human plasma. In this way, most of the contaminated heparin samples did not meet the novel potency limit of 180IU/mg. However, also more than 50% of the uncontaminated samples had <180IU/MG. In contrast to the aPTT, the PT specifically detects OSCS and other heparin mimetics (LOD 3%). About ten times more sensitive are both the Inc-PolyH-assay and the Inc-aXa-assay, two rapid and simple quantification assays for heparin mimetics. The determined OSCS contents of the heparin samples excellently correlated with those calculated from the NMR spectra. In conclusion, NMR proved to be the current spectroscopic method of choice. The two two-step-assays represent options to supplement NMR, especially as tests for the initial screening, since they detect any heparin mimetic without requiring special expertise for interpretation of the results.

Development and validation of a liquid chromatography/mass spectrometry method for pharmacokinetic studies of OZ78, a fasciocidal drug candidate

Fascioliasis is a zoonotic disease of considerable public health and great veterinary significance and new drugs are needed. OZ78 is a promising fasciocidal drug candidate. In order to support the development of OZ78, including pharmacokinetic (PK) studies an accurate, precise, and selective liquid chromatography/mass spectrometry (LC/MS) method for OZ78 was developed for sheep plasma and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. Protein precipitation was used for sample clean up. Separation was performed through a Phenomenex C8(2) analytical column (50.0 mm × 2.0 mm, 5 μm) with a mobile phase of acetonitrile (buffer B) and 5 mM ammonium formate (buffer A) at a flow-rate of 0.3 mL/min and a gradient from 20% to 95% acetonitrile. The mass spectrometer was operated under selected ion monitoring, and orifice voltage set to −4.1 kV and ion spray temperature to 400 °C. Nitrogen was used as a nebulizer, curtain, and collision gas. OZ78 was monitored at 321.4 m/ z (deprotonated parent compound, M-). The validated linear dynamic range was between 156.25 ng/mL and 5 μg/mL and the achieved correlation coefficient ( r 2) was greater than 0.99. The validation results demonstrated that the developed LC/MS method is precise, accurate, and selective for the determination of OZ78 in sheep plasma. The method was successfully applied to the evaluation of the PK profile of OZ78 in sheep.

Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test

BackgroundThe ideal identification of Staphylococcus aureus clinical isolates requires a battery of tests and this is costly in resource limited settings. In many developing countries, the tube coagulase test is usually confirmatory for S. aureus and is routinely done using either human or sheep plasma. This study evaluated Mannitol salt agar and the deoxyribonuclease (DNase) test for improving the efficiency of the tube coagulase test in resource limited settings. The efficiency of human and sheep plasma with tube coagulase tests was also evaluated.MethodsOne hundred and eighty Gram positive, Catalase positive cocci occurring in pairs, short chains or clusters were subjected to growth on Mannitol salt agar, deoxyribonuclease and tube coagulase tests. Of these, isolates that were positive for at least two of the three tests (n = 60) were used to evaluate the performance of the tube coagulase test for identification of S. aureus, using PCR-amplification of the nuc gene as a gold standard.ResultsHuman plasma was more sensitive than sheep plasma for the tube coagulase test (sensitivity of 91% vs. 81% respectively), but both plasmas had very low specificity (11% and 7% respectively). The sensitivity and specificity of the tube coagulase test (human plasma) was markedly improved when Mannitol salt agar and DNase were introduced as a tri-combination test for routine identification of Staphylococcus aureus (100% specificity and 75% sensitivity). The specificity and sensitivity of Mannitol salt agar/DNase/tube coagulase (sheep plasma) combination was 100% and 67%, respectively.ConclusionThe efficiency of the tube coagulase test can be markedly improved by sequel testing of the isolates with Mannitol salt agar, DNase and Tube coagulase. There is no single phenotypic test (including tube coagulase) that can guarantee reliable results in the identification of Staphylococcus aureus.

Composition of OSCS-contaminated heparin occurring in 2008 in batches on the German market

In 2008, some 900 cases of adverse events associated with the use of heparin were reported to the Food and Drug Administration of USA and the Federal Institute of Drugs and Medical Devices in Germany. 238 patients died from heparin in the USA. In March 2008, oversulfated chondroitin sulfate (OSCS) was identified to be responsible for these cases. NMR spectroscopic evaluation of heparin samples revealed OSCS, dermatan sulfate (DS), chondroitin sulfate A and C as well as various residual solvents to be present in heparin batches, which could not be identified by means of conventional methods described in various pharmacopoeias at that time. In order to evaluate the situation on the German market, 145 representative samples were collected in 2008 and analyzed by means of 1H NMR spectroscopy, water determination, optical rotation and sheep plasma clotting assay. 66 samples were found to contain pure heparin, 51 samples heparin plus DS, 5 samples heparin plus OSCS, and 23 samples heparin, DS and OSCS, each in varying amounts. In 94 out of 145 batches especially ethanol was found in strongly varying amounts up to about 9.5%. Traces of acetone and formic acid were found with concentrations up to 0.04%, as well as sodium acetate and methanol up to 0.5%. Additionally, in many batches the content of water was found to be relatively high. Whereas the optical rotation was able to identify samples with a high contamination of OCSC, all samples tested fulfilled the requirements of the anticoagulation potency assay of the European Pharmacopoeia 6.0. The presented analysis of a representative set of heparin samples proves the suitability of 1H NMR spectroscopy for the quality control of heparin of both glycosaminoglycans and residual solvents.

Measurement of monoterpenes and sesquiterpenes in serum, plasma, and rumen fluid from sheep

Studies involving the consumption, metabolism, and elimination of terpenes by small ruminants consuming terpene-laden shrubs as well as those exploring the potential for natural products as rumen modifiers could benefit from a procedure that measures terpenes in both blood and rumen fluid and that is suitable for either serum or plasma. The objective of this study was to modify an existing procedure for plasma utilizing solid phase extraction/gas chromatography, and extend its use for measurement of structurally diverse mono- and sesquiterpenes in three fluids (serum, plasma, and rumen fluid) from sheep. Generally, terpene recovery was lower from rumen fluid than from serum or plasma, although the extent and direction of differences varied among chemicals. Fourteen terpenes (camphene, β-pinene, α-terpinene, p-cymene, cis-β-ocimene, 1,8-cineole, γ-terpinene, terpinolene, linalool, camphor, longifolene, β-caryophyllene, α-humulene, and caryophyllene oxide) were recovered from serum at near unity. Recovery from rumen fluid was lower than that for serum or plasma for most terpenes, but eight ( p-cymene, 1,8-cineole, cis-sabinene hydrate, terpinolene, borneol, terpin-4-ol, α-terpineol, and caryophyllene oxide) were recovered at near unity. Yet, 15 terpene recoveries were below 0.75 ng/ng (tricyclene, α-pinene, camphene, sabinene, β-pinene, myrcene, 2-carene, 3-carene, α-terpinene, cis-β-ocimene, limonene, γ-terpinene, longifolene, β-caryophyllene, and α-humulene). Oxygenated monoterpenes were typically recovered in greater quantities and hydrocarbon monoterpenes were least effectively recovered with this method. This procedure allows for simultaneous measurement and recovery adjustment of a number of terpenes from serum, plasma, and rumen fluid of sheep.

Relationship between Grazing Lamb Growth Rate and Blood Plasma Analytes as Profiled by Gas Chromatography with Time-of-Flight Mass Spectrometry (GC-TOF/MS).

There can be considerable variation in the performance of individual lambs grazing on the same pasture. Gas chromatography with time-of-flight mass spectrometry (GC-TOF/MS) was used to profile the relative abundances of metabolites in plasma from growing lambs to determine any correlation effects between plasma metabolites and liveweight gain. Analysis of relative abundance of 336 analyte clusters and liveweight gain revealed that the growth rates of female lambs were significantly positively correlated with 5 analyte clusters and negatively correlated with 5 other analyte clusters. Growth rates of male lambs were likewise significantly positively correlated with 9 analyte clusters and negatively with 5 analyte clusters. Analytes identified as being associated with lamb growth rate included the amino acids valine, methionine, phenylalanine, cystine and asparagine, and oxalic acid, phenylacetic acid, and phosphoric acid. A number of currently unidentified analytes were significantly correlated with growth rate. Stepwise regression of the analytes on lamb growth rate yielded relationships that accounted for 48% and 58% of the variation in female and male lamb growth rates, respectively. This study demonstrated that by using GC-TOF/MS in combination with multivariate statistical techniques it is possible to correlate the presence of specific analytes in sheep plasma with growth rate.

Simple liquid chromatography method for the quantification of irinotecan and SN38 in sheep plasma: Application to in vivo pharmacokinetics after pulmonary artery chemoembolization using drug eluting beads

A rapid and simple liquid chromatography–fluorescence detection (LC–FD) method was developed and validated for the simultaneous quantification of irinotecan (CPT11) and SN38 in sheep plasma. Camptothecin (CPT) was used as the internal standard. A single step protein precipitation with acetonitrile was used for sample preparation. The separation was achieved using a 5 μm C18 column (250 mm × 4.5 mm, 5 μm) with a mobile phase composed of 36 mM sodium dihydrogen phosphate dehydrate and 4 mM sodium 1 heptane sulfonate–acetonitrile (72:28), the pH of the mobile phase was adjusted to 3. The flow rate was 1.45 mL/min and the fluorescence detection was operated at 355/515 nm (excitation/emission wavelengths). The run time was 13 min. The method was validated with respect to selectivity, extraction recovery, linearity, intra- and inter-day precision and accuracy, limit of quantification and stability. The method has a limit of quantification of 5 ng/mL for both CPT11 and SN38. The assay was linear over concentrations ranging from 5 to 5000 ng/mL and to 240 ng/mL for CPT11 and SN38, respectively. This method was used successfully to perform plasma pharmacokinetic studies of CPT11 after pulmonary artery embolization (PACE) in a sheep model. It was also validated for CPT11 and SN38 analysis in sheep lymph and human plasma.

Assessment of a sheep animal model to optimise photodynamic therapy in the oesophagus

Precursor lesions of oesophagus adenocarcinoma constitute a clinical dilemma. Photodynamic therapy (PDT) is an effective treatment for this indication, but it is difficult to optimise without an appropriate animal model. For this reason, we assessed the sheep model for PDT in the oesophagus with the photosensitiser meta-(tetra-hydroxyphenyl) chlorin (mTHPC). Twelve sheep underwent intravenous mTHPC injection, blood sampling and fluorescence measurements. mTHPC's pharmacokinetics was measured in vivo and in plasma by fluorescence spectroscopy. Biopsies of sheep oesophagus were compared to corresponding human tissue, and the mTHPC's biodistribution was studied under fluorescence microscopy. Finally, the sheep oesophageal mucosa was irradiated, 4 days after mTHPC's injection. Histologically, the sheep and human oesophagus were closely comparable, with the exception of additional fatty tissue in the sheep oesophagus. mTHPC's pharmacokinetics in sheep and human plasmas were similar, with a maximum of concentration in the sheep 10 hours after i.v. injection. mTHPC's pharmacokinetics in vivo reached its maximum after 30-50 hours, then decreased to background levels, as in humans under similar conditions. Two days after injection, mTHPC was mainly distributed in the lamina propria, followed by a penetration into the epithelium. The sheep and human tissue sensitivity to mTHPC PDT was similar. In conclusion, this model showed many similarities with humans as to mTHPC's plasma and tissue pharmacokinetics, and for tissue PDT response, making it suitable to optimise oesophagus PDT.

Metabolic fate of fatty acids involved in ruminal biohydrogenation in sheep fed concentrate or herbage with or without tannins1

A 2 x 2 factorial experiment was carried out to evaluate the effect of herbage or concentrate feeding and dietary tannin supplementation on fatty acid metabolism and composition in sheep ruminal fluid, plasma, and intramuscular fat. Twenty-eight male lambs were divided into 2 equal groups at 45 d of age and kept in individual pens. One group was given exclusively fresh herbage (vetch), and the other group was fed a concentrate-based diet. Within each treatment, one-half of the lambs received supplementation of quebracho powder, providing 4.0% of dietary DM as tannins. Before slaughter, blood samples were collected. The animals were slaughtered at 105 d of age, and ruminal contents and LM were collected. Blood plasma, ruminal fluid, and LM fatty acid composition was determined by gas chromatography. Tannin supplementation reduced (P < 0.05) the concentration of stearic acid (-49%) and increased the concentration of vaccenic acid (+97%) in ruminal fluid from concentrate-fed lambs. Within concentrate- and herbage-based diets, tannin supplementation reduced the accumulation of SFA in blood (P < 0.05) compared with lambs fed the tannin-free diets. When tannins were included in the concentrate, the LM contained 2-fold greater concentrations of rumenic acid compared with the LM of the lambs fed the tannin-free concentrate (0.96 vs. 0.46% of total extracted fatty acids, respectively; P < 0.05). The concentration of PUFA was greater (P < 0.05) and SFA (P < 0.01) less in the LM from lambs fed the tannin-containing diets as compared with the animals receiving the tannin-free diets. These results confirm, in vivo, that tannins reduce ruminal biohydrogenation, as previously reported in vitro. This implies that tannin supplementation could be a useful strategy to increase the rumenic acid and PUFA content and to reduce the SFA in ruminant meats. However, the correct dietary concentration of tannins should be carefully chosen to avoid negative effects on DMI and animal performance.

Genetic polymorphism in sheep plasma detected using antibodies to human plasminogen

Protein variation was identified in sheep when Western blots of polyacrylamide gels (routinely used to resolve transferrin polymorphism) were stained using antibodies to human plasminogen. The affinity of the antibodies to ovine plasma was less than 7% that of a human standard but they bound specifically to a single polymorphic protein. In 146 lambs and their parents the inheritance of the ovine plasminogen antigen polymorphism was consistent with four autosomal alleles segregating codominantly. However, an additional two lambs had types which were incompatible with their putative parents. The pedigrees of these lambs were tested by DNA fingerprinting and shown to have been incorrectly recorded. The genetic polymorphism detected by human plasminogen antiserum provided a probability of sire exclusion (PE) ranging from 0.04 to 0.32 and a polymorphic information content (PIC) of 0.08 to 0.50 in flocks of five sheep breeds: Perendale, Romney, Merino, Texel and Coopworth (in order of increasing genetic variation in this locus). Significant differences in allele frequency were observed between breeds but sampling did not assess the variation among flocks within a breed.