An automated micro-method for the enzymatic determination of D-B-hydroxybutyrate in ruminant plasma

An enzymatic 'reaction rate' micro-method for the rapid routine estimation of D-B-hydroxybutyrate (D-B- HOB ) in ruminant plasma, using an I.L. Multistat III centrifugal analyzer, is described. Reaction conditions were optimized to give a linear response for plasma D-B- HOB concentrations between 100 and 2500 mumoles per litre, at 30 degrees C and pH 9.0. For the standardized method the within-run and between-run coefficients of variation for deproteinised ovine plasma were consistently less than 3.5%. There was good agreement between plasma concentrations obtained by the present method and both original U.V. end-point technique (r = 0. 927b = 0.950) and a colorimetric end-point procedure (r = 0.937. b = 0.879). Untreated ovine and bovine plasma consistently exhibited high 'blank' activity and this was directly correlated with plasma lactate dehydrogenase (LDH) activity in both species (r = 0.971; p less than 0.001 and r = 0.949; p less than 0.001 respectively). The distribution of LDH activity in man was similar to sheep but, contrastingly , non-specific interference was extremely low in human plasma and unrelated to LDH. Horse, chicken and rat had negligible 'blank' activity and comparatively low LDH levels. In both cattle and sheep non-specific interference was abolished by perchloric acid precipitation. In the sheep subtraction of 'blank' activity gave D-B- HOB concentrations for untreated plasma comparable to those in deproteinised samples. However, in the bovine, D-B- HOB levels remained significantly (t = 6.44; p less than 0.001) higher even after 'blank' correction. In contrast to man and other non-ruminants, perchloric acid precipitation is essential in ruminants to avoid false overestimation of plasma D-B- HOB levels. Plasma with EDTA as anticoagulant and serum gave concentrations of D-B- HOB approximately 60% lower, than samples containing heparin or oxalate/fluoride. However, heparin was associated with much higher (up to 50%) non-specific NAD reduction than oxalate/fluoride. High levels of acetoacetate (400-1000 mumoles per litre) reduced the recovery of D-B- HOB from ovine plasma by less than 10%. This effect was negated by the inclusion of hydrazine hydrate in the reaction mixture. Perchlorate ion concentrations above 25 mumoles per litre per test dramatically inhibited the assay in ovine plasma, and therefore precipitation conditions must be carefully controlled. Plasma with oxalate/fluoride as anticoagulant showed the greatest stability in storage; 24 hours at room temperature, one week at +4 degrees C and at least one month at -20 degrees C.     

Pipecolic acid in rumen fluid and plasma in ruminant animals

A survey was conducted to investigate the physiological levels of pipecolic acid (Pip) in rumen fluid and plasma of ruminants such as goats and cattle in the presence or absence of rumen protozoa. The concentration of Pip was determined using HPLC. Basal Pip levels in the rumen fluid and plasma of normal faunated animals were 21 ± 8 and 2.3 ± 1.3 µM, respectively, and levels increased 1–2 h after feeding. The Pip levels in the rumen fluid and plasma of faunated goats and cattle were significantly higher than those of defaunated goats and unfaunated cattle. A small amount of Pip was also found in the rumen fluids of the defaunated and unfaunated animals; this appeared to be derived from feeds such as hay cube and corn silage. The results obtained in the present study suggest that a significant amount of rumen‐produced Pip is likely to be absorbed into the plasma of the host animals and that rumen protozoa significantly enhance the concentration of Pip in the rumen fluid and plasma of ruminant animals.     

烟酸在反刍动物脂肪代谢中的研究进展

烟酸是动物机体的必需维生素,是合成机体需要的重要辅酶NAD和NADP的直接前体,对动物碳水化合物、蛋白质、脂类代谢有重要影响。文中阐述了烟酸的理化性质及其在脂肪代谢中的作用、烟酸对瘤胃中挥发性脂肪酸的影响及对反刍动物脂肪代谢的调控等,以期促进烟酸在反刍动物脂肪代谢中的研究。     

Assessment of three automated assays for C-reactive protein determination in dogs

OBJECTIVE: To determine the characteristics of an automated canine C-reactive protein (CRP) assay and evaluate 2 human CRP assays for use in dogs. Animals-56 client-owned dogs with pyometra and 11 healthy control dogs. PROCEDURES: Samples from 11 dogs with high (> 100 mg/L) or low (< 10 mg/L) CRP concentrations (determined by use of a canine ELISA) were evaluated by use of the automated canine CRP assay. Intra- and interassay imprecision was determined (by use of those 2 plasma pools), and assay inaccuracy was assessed by use of logistic regression analysis of results obtained via ELISA and the automated canine CRP assay. Two automated human CRP assays were used to measure plasma CRP concentration in 10 dogs. RESULTS: By use of the ELISA, mean +/- SD plasma CRP concentration was 96.1 +/- 38.5 mg/L and 10.1 +/- 23.2 mg/L in dogs with pyometra and control dogs, respectively. The automated canine assay had intra-assay coefficients of variation (CVs) of 7.8% and 7.9%, respectively, and interassay CVs of 11.1% and 13.1%, respectively. Results from the automated assay were highly correlated with results obtained via ELISA. The human assay results did not exceed 0.4 mg/L in any dog. CONCLUSIONS AND CLINICAL RELEVANCE: The automated canine CRP assay had less interassay imprecision, compared with the ELISA. The 2 human CRP assays were not suitable for analysis of canine plasma samples. The automated canine CRP assay was more precise than the ELISA for serial evaluations of plasma CRP concentration in dogs.     

An automated experimental milker for rat

The development of sucking pressure was investigated with an artificial nipple in 16 male and female rat pups on postnatal days 4, 7, 10, 14, and 18. The rat pups refused to suck on the artificial nipple on postnatal day 14 or day 18 thus negative sucking pressure had to be calculated by regression analysis. As a result, the mean maximum intra-oral negative sucking pressure on day 18 was calculated to be -160.1 mmHg in the male and -103.4 mmHg in the female. Based on these results, the maximum level of negative pressure of the automated experimental rat milker was set at -160 mmHg. The automated experimental milker for rat is able to collect milk from lactating mothers by alternating negative and atmospheric pressures through two solenoid valves and a vacuum pump attached to a microcomputer. Mother rats were milked with the automated experimental milker on postpartum days 4, 7, 10, 14 and 18 of a single lactation period. The maximum mean milk yield with this machine was 3.18 +/- 1.37 g, obtained on day 14 of the lactation period. This quantity is considerably lower than previously reported values obtained by measuring differences in body weight of the offspring and mother rat before and after suckling. It is necessary to further optimize this system, but the milk yield in the present study is adequate for chemical analysis.     

An improved fluorimetric determination of alpha-mannosidase activity in bovine plasma.

This paper describes a manual fluorimetric method for the assay of acidic alpha-mannosidase activity in bovine plasma. The optimum conditions for the assay of this enzyme were studied. The assay method devised includes the addition of zinc to the substrate, which stimulates activity by approximately twofold, and reduces the optimum substrate concentration. This latter feature affords considerable cost saving in each test. We have also shown that the alpha-mannosidase activity in lithium heparin plasma, EDTA plasma and blood serum is the same whether the plasma/serum is separated from the cells/clot at 6, 20 or 25 hours after sample collection. This has eliminated the previous necessity of having to deliver whole blood samples to the laboratory within 6 hours of collection. Furthermore samples for the supplementary neutrophil assay can now be taken at the same time as those for the plasma test, and both samples forwarded together. The plasma alpha-mannosidase assay is a rapid and reliable screening test for the mannosidosis genotype and for detecting carrier animals. Carrying out this plasma assay in conjunction with the more definitive neutrophil assay provides a reliable method of distinguishing homozygotes and heterozygotes from normal animals.     

An automated method for the measurement of methaemoglobin in avian blood

An automated method has been developed for the measurement of methaemoglobin (metHb) in blood using a biochemistry analyser. The method was validated using blood collected from red-legged partridges exposed in vitro to increasing concentrations of nitrite in order to obtain different percentages of metHb. Results obtained using the original manual method and those using the new automated technique were compared and no significant differences were found. Intra-day and inter-day variabilities (8.8% and 2.6%, respectively) were acceptable for samples containing high levels (63-81%) of metHb. Methaemoglobin measured in blood samples stored in liquid nitrogen was stable for 10 days, but increased significantly by day 20 in nitrite-treated samples.     

HPLC法检测家兔血浆中地克珠利含量

建立了快速、稳定的家兔血浆中地克珠利提取方法,为进一步研究家兔组织中地克珠利的残留量提供基础。采用HPLC法以妥曲珠利为内标,N,N-2甲基甲酰胺及乙腈联合沉淀法提取家兔血浆中地克珠利,色谱柱为Hypersil ODS（2.5μm,250 mm×4.6 mm）,流动相为乙腈∶水∶三乙胺∶冰乙酸=55∶45∶0.2∶0....     

Evaluation of an enzymatic immunoassay for free thyroxine determination in canine serum

Free thyroxine (FT4) and cholesterol were measured in 400 dogs with either suspected hypothyroidism or dermatological signs such that hypothyroidism needed to be ruled out. Hypothyroidism was diagnosed in 68 dogs from the history, physical examination and stated lower reference limit (<7 pmol/L) for FT4 in euthryoid dogs. Dogs with FT4 concentrations in the range 6–9 pmol/L were finally categorized as hypo- or euthyroid either on the basis of retesting after 2 months or on their clinical response to thyroid replacement therapy over at least 2 months.The enzyme immunoassay evaluated in this paper is considered to be of clinical value and offers many advantages compared with radioimmunoassays.Abbreviations FT4 free thyroxine fraction - MEIA microparticle enzyme immunoassay - RIA radioimmunoassays - T4 thyroxine - TBG thyroxine-binding globulin     

An update of a dynamic model of ruminant digestion

The progressive development of a dynamic model of ruminant digestion for evaluation of factors affecting nutritive value of feedstuffs is outlined. Significant changes have been introduced in many of the interactive subunits that accommodate the digestion of 12 chemical constituents (soluble carbohydrate, organic acids, starch, pectin, hemicellulose, cellulose, lipids, soluble protein, insoluble protein, nonprotein N, lignin and ash), microbial growth, animal interactions and summary computations. Explicit consideration of soluble carbohydrates, organic acids and pectins has been replaced by an aggregate fraction. A section has been added to follow dynamic aspects of volatile fatty acid metabolism in the rumen. Evaluations of the model were made by comparing its behavior with that observed experimentally for sheep fed a number of diets. These included an alfalfa diet fed at two intake levels either hourly or once daily, a dried subterranean clover diet fed hourly, a forage oat diet fed every 3 h and an 80% concentrate diet fed twice daily. For frequently fed diets, model estimates, in general, agreed very well with experimental estimates. Although changes in representations of protein metabolism were helpful, the pool size of ruminal ammonia N was not simulated well. Digestion and passage in the model occurred more rapidly than was observed when animals were fed high-quality forages once daily and rates of particle size reduction, fermentation and passage were lower than observed when low-quality diets were fed. It was concluded that the model can serve as a useful and adaptable tool for analyzing factors affecting nutritive value. Particle size reduction and passage from the rumen, dynamics of protein metabolism and utilization, and water dynamics were identified as areas requiring further research.     

Fuel homeostasis in the ruminant

Sufficient amounts of energy are stored in the animal body to support its needs during periods when dietary intake does not meet energy requirements. To be utilized, these energy reserves must be converted to compounds appropriate for oxidation at the cellular level. In addition, energy supplies must be transferred from storage sites to sites of utilization. The biochemical pathways of oxidation provide not only a means of deriving energy from carbon compounds, but also a means of transferring carbons from one type of energy source to another. The transfer of carbons between carbohydrates, lipids, ketone bodies, and proteins is regulated by endocrine and substrate effects. Regulation of this activity constitutes fuel homeostasis. Breakdowns in these homeostatic mechanisms result in metabolic disease.     

Comparative plasma ampicillin levels and bioavailability of five parenteral ampicillin formulations in ruminant calves

Summary

Plasma ampicillin concentrations were determined in an eight‐ways crossover trial involving six ruminant calves, which were treated intravenously (i.v.) with sodium ampicillin at 15.5 mg/kg and intramuscularly (i.m.) with five different ampicillin trihydrate or ampicillin anhydrate formulations at 7.7 mg/kg. The mean plasma concentration‐time curve (C_p)after intravenous ampicillin sodium administration was described biexponentially, as: C_p = 38.8 e ‐0.0268t + 0.45 e ‐0.0058t.

Intramuscular injection, into the lateral neck, of Ampikel‐20^® and Polyflex^® resulted in 100 per cent bioavailabilities within 12 h post injection (p.i.), but the biological half‐lives (t½>) were different, being 2.1 and 3.8 h, respectively. Ampikel‐20^® produced the hïghest peak plasma drug concentrations (mean C _max:4.8 μg ampicillin/ml). After intramuscular injection of Penbritin® the mean bioavailability for the first 12 h p.i. was 63 per cent, the mean t½>, was 5.9 h, and the mean C_max was 1.8 μg/ml. Treatment with Albipen^® and Duphacillin^® resulted in low plasma ampicillin levels, which were maintained for 3 to 6 days p.i., limited bioavailability during the first 12 h p.i., and a mean t½> of 22.2 and 11.9 h, respectively. Plasma concentrations of ampicillin from four hours onwards after i.m. and s.c. administration of Ampikel‐20^® at a dose level of 15.5 mg/ kg were similar.

The duration of potentially therapeutic plasma ampicillin concentrations after administration of each formulation is presented. Pre‐slaughter withdrawal times for diseased calves are suggested for the different formulations studied.     

Characterization of the caprine model for ruminant brucellosis

The relationship between man, the goat, and brucellosis is historical. Today Brucella melitensis and Brucella abortus pose a serious economic and public health threat in many countries throughout the world. Infection of pregnant goats and sheep with B. melitensis results in abortion during the third trimester of pregnancy. Although nearly eradicated in the US, bovine brucellosis is still a problem in many countries and the potential for re-infection of domestic stock from wildlife reservoirs in this country is a regulatory nightmare. Humans infected with this pathogen develop undulant fever, which is characterized by pyrexia, arthritis, osteomyelitis, and spondylitis. Although available for both organisms, currently available vaccines have problems ranging from false positive serological reactions to limited efficacy in different animal species. With the continued need for new and better vaccines, we have further developed a goat model system to test new genetically derived strains of B. melitensis and B. abortus for virulence as measured by colonization of maternal and fetal tissues, vaccine safety, and vaccine efficacy.     

Evaluation of an automated spectrophotometric assay for the determination of total sialic acid in canine serum

This study validates an automated enzymatic assay using the Cobas Fara (Roche) centrifugal analyser, which offers a reliable measurement of the total sialic acid concentration in canine serum as assessed by evaluating the precision and accuracy. Data are presented on the biological variation in the total serum sialic acid concentration. Measurements of total serum sialic acid concentration appear to be useful in distinguishing dogs with neoplastic disorders from clinically healthy dogs.Abbreviations AcNeu N-acetylneuraminic acid - CV% coefficient of variation - LASA lipid-associated sialic acid - S ² component of variance     

犬血浆中特比萘芬的高效液相色谱检测方法

建立并优化一种简单、快速、灵敏、高选择性的HPLC方法测定犬血浆中特比萘芬血药浓度,并考察方法的稳定性。特比萘芬血浆样品经提取剂乙腈-异丙醇(用磷酸调p H至3.0)(40:60,V/V)液-液萃取后,提取液经40℃氮气吹干,用流动相复溶后离心过滤,取上清液20μL进样分析,对血样处理后及标准溶液保存不同时间后的稳定性进行了考察。色谱柱Agilent 5 HC-C18(2)柱(250 mm×4.6 mm,5μm);流动相甲醇-0.1%磷酸水(59:41,V/V);流速0.8 m L/min;紫外检测波长223 nm;柱温35℃。色谱峰分离良好,无干扰。特比萘芬最低检测浓度为0.01μg/m L,线性范围是0.02~5μg/m L,相关回归方程:y=356.28x-2.0311(r2=0.99989)。低、中、高血浆质控样品日内RSD均小于5%,日间RSD均小于7%,方法平均回收率分别为108.9%,100.4%,102.1%。血样处理后8 h内样品能保持较好稳定性,血样处理后3次循环冻融能保持较好稳定性。血样处理后于-20℃条件下冻存能够保持稳定2个月,标准溶液有效保存期同样为2个月。本研究建立的特比萘芬血样浓度测定方法有较高的准确度和灵敏度,适用于候选化合物的体内药代动力学研究。