The suitability of a faecal suspension of sheep as inocula for the estimation of utilizable crude protein of feeds by in vitro incubation

The objective of the experiments was to study the suitability of using a faecal suspension of sheep for the estimation of the utilizable crude protein (uCP) of feeds for sheep by an in vitro incubation. Twenty-four single feeds and eight feed mixtures were used as incubation substrates. In Experiment 1, the gas production after the in vitro incubation with rumen fluid or with a faecal suspension of a sheep were compared using the Hohenheim gas test. It was found that there were significant linear regression between the 24, 48 and 72 h gas production with rumen fluid and those with faecal suspensions of 35, 50, 100 and 150 g wet faeces of sheep (which were 18.6, 23.5, 52.0 and 70.5 g faeces DM, respectively) per litre McDougall's buffer (P < 0.0001). The highest regression coefficient (r2) was calculated between the gas production after inoculation with a suspension of 100 g wet faeces per litre McDougall's buffer (x, ml x 200 mg (-1) feed DM) for 48 h and the gas production after inoculation with rumen fluid (y, ml x 200 mg (-1) feed DM) for 24 h: y = 0.82 (+/- 0.07)x + 9.87 (+/-3.83), r2 = 0.82, n = 32, P < 0.0001. Based on these results, in Experiment 2 the estimation of utilizable crude protein (uCP) of feeds was compared by using the in vitro incubation technique of Zhao and Lebzien (2000), where feeds were inoculated either with rumen fluid or with a faecal suspension (100 g wet faeces of sheep, i.e. 52 g faeces DM per litre McDougall's buffer). The results indicated that there were no significant differences of the estimated uCP after inoculation with rumen fluid or the faecal suspension (P > 0.05). A significant regression was found between the uCP after incubation for 48 h with 100 g wet faeces (x, g x kg (-1) DM) and the uCP after incubation for 24 h with rumen fluid (y, g x kg(-1) DM): y = 0.95 (+/-0.10)x - 4.90 (+/-26.70), r2 = 0.75, n = 32, Although this regression was significant, the coefficient r2 was not high. Therefore, further research is needed before sheep faeces could replace rumen fluid as an inocula for the estimation of uCP by the in vitro incubation technique.     

Study on the interaction between protein and Eu(III)-chlorotetracycline complex and the determination of protein using the fluorimetric method.

Chlorotetracycline (CTC) can react with europium ions Eu3+, and the complex emits the intrinsic fluorescence of Eu3+. The intensity is greatly enhanced by proteins and this forms the basis of a new fluorimetric method for determination of protein. Further research indicates that under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of proteins, in the range 2.0 x 10(-7)-1.0 x 10(-5) g/mL for bovine serum albumin (BSA) (linear equation, I(f) = 34.35933 + 11.54467 x 10(6)C)(r = 0.99895) and 8.0 x 10(-7)-1.0 x 10(-5) g/mL for human serum albumin (HSA) (linear equation, I(f) = 76.58881 + 5.3569 x 10(6)C) (r = 0.99283). Detection limits (S/N = 3) were 8.9 x 10(-9) g/mL for BSA and 3.3 x 10(-8) g/mL for HSA. In an assay for BSA in calf serum, this method gave a value close to that determined by the UV spectrophotometric method.     

Interpretation of Serum Calcium in Patients with Abnormal Serum Proteins

Two hundred consecutive specimens received in this laboratory for “liver function tests” showed a wide range of abnormal protein concentrations. Calcium concentration correlated closely with albumin (r = 0·867) but less closely with total protein (r = 0·682). A simple formula for adjusting calcium concentration was derived from the regression equation of calcium on albumin. Adjusted calcium = calcium - albumin + 4·0, where calcium is in mg/100 ml and albumin in g/100 ml.Low calcium concentrations were found in 49 (24·5%) and raised concentrations in six (3%) of the 200 blood specimens taken for liver function tests. After adjustment, the 95% limits of the observed range were identical with the 95% limits of the normal range determined in this laboratory. Unlike adjustments based on total protein or specific gravity, the adjustment on albumin in 39 specimens which showed hypergammaglobulinaemia on electrophoresis gave normal calcium concentrations.     

Clinical usefulness of estimation of serum fructosamine concentration as a screening test for diabetes mellitus.

Fructosamine, a putative measure of serum glycosylated proteins, was measured in 74 subjects referred for oral glucose tolerance tests. A normal range (mean (2 SD] of 1.6 (0.4) mmol/l (40(10) mg/100 ml) derive from results obtained in 83 healthy non-diabetic volunteers permitted the detection of 15 out of 17 (88%) subjects with proved diabetes and yielded only five (9%) false positive diagnoses. Fructosamine concentrations correlated significantly (p less than 0.001) with fasting plasma glucose concentrations (r = 0.76) and glycosylated haemoglobin concentrations (r = 0.70). A longitudinal study suggested that fructosamine concentration was an index of intermediate term (one to three weeks) blood glucose control. Fructosamine concentration was not related to uraemia and did not depend on albumin or total protein concentrations, provided that serum albumin concentrations remained above 30 g/l. Estimation of fructosamine concentrations is a fully automated procedure and may provide a simple means of screening for diabetes mellitus.     

Effect of body orientation on regional lung expansion in dog and sloth

Recent studies (E.A. Hoffman, J. Appl. Physiol. 59: 468-480, 1985) using fast multisliced X-ray computed tomography have demonstrated a ventral-dorsal gradient of fractional lung air content (3.29% air/cm lung height) in supine dogs and an essentially uniform ventral-dorsal air content distribution in the prone dogs [mean = 66 +/- 0.6% (SE) air content]. Since the prone orientation is the dog's normal body posture, we sought to study an animal whose normal body posture was "opposite" to that of the dog. Four two-toed sloths were scanned in the Dynamic Spatial Reconstructor in the prone and supine postures. A supine fractional air content gradient was demonstrated with a regression equation of y = 2.09x + 74.3 (r = 0.92), where y is percent air content and x is vertical height in the lung, and ventral-dorsal air content distribution in the prone posture was uniform with a mean of 85 +/- 0.4% (SE) air content. The low functional residual capacity lung density in the sloth was attributable to unusually large alveoli. The mean heart volume-to-body weight ratio in the dogs was 16.4 +/- 0.6 (SE) ml/kg and that in the sloth was 7.3 +/- 0.4 (SE) ml/kg. Mean lung volume-to-body weight ratios for dogs and sloths were 57 +/- 7 (SE) and 89 +/- 6 ml/kg, respectively. Of particular interest was the fact that large changes in prone vs. supine rib cage and diaphragm geometry previously found in dogs did not occur in sloths, though significant alterations of ventral and dorsal lung geometry prone vs. supine were demonstrated, and lung shape changes in both dog and sloth are attributable to shifts in the intrathoracic position of mediastinal structures.     

Measurement of interstitial albumin in human skeletal muscle and adipose tissue by open-flow microperfusion

The absolute concentration of albumin was measured in the interstitial fluid of subcutaneous adipose tissue and skeletal muscle in six healthy volunteers by combining the method of open-flow microperfusion and the no-net-flux calibration technique. By use of open-flow microperfusion, four macroscopically perforated double lumen catheters were inserted into the tissue regions of interest and constantly perfused. Across the macroscopic perforations of the catheters interstitial fluid was partially recovered in the perfusion fluid. Catheters were perfused with five solutions, each containing different concentrations of albumin. Absolute interstitial albumin concentrations were calculated by applying linear regression analysis to perfusate vs. sampled albumin concentration (no-net-flux calibration technique). Interstitial albumin concentrations were significantly lower (P < 0.0001) in adipose tissue (7.36 g/l; r = 0.99, P < 0.0003; range: 4.3-10.7 g/l) and in skeletal muscle (13.25 g/l; r = 0.99, P < 0.0012; range: 9.7 to 15.7 g/l) compared with the serum concentration (48.9 +/- 0.7 g/l, mean +/- SE, n = 6; range: 46.4-50.4 g/l). Furthermore, interstitial albumin concentrations were significantly higher in skeletal muscle compared with adipose tissue (P < 0.01). The study indicates that open-flow microperfusion allows stable sampling of macromolecules from the interstitial space of peripheral tissue compartments. Moreover, the present data report for the first time in healthy humans in vivo the true albumin concentrations of interstitial fluid of adipose tissue and skeletal muscle.     

染料结合法测定荞麦种子蛋白质含量的研究

以考马斯亮蓝G250为染料,结合经典的凯氏定氮法测定结果,对影响染料结合法测定蛋白质含量的振荡时间、温度、考马斯亮蓝溶液浓度等因素进行了研究,并分析了由考马斯亮蓝染料和蛋白质结合后染料结合量(OD值差)与凯氏法测得的蛋白质百分含量之间的相关性。结果表明:测定的适宜条件是:温度15℃,处理时间50min,考马斯亮蓝溶液的浓度是0.06mg/mL。此条件形成的络合物较稳定,重复性较好,并且所测的染料结合量与凯氏定氮法测得的蛋白质含量间呈极显著的一元线性回归和相关关系。栽培甜荞和栽培苦荞的回归方程分别为:y=15.364x+3.865和y=10.769x+6.287,这两个回归方程差异显著,不能合并,分别适合于快速估计甜荞和苦荞种子的蛋白质含量。     

Enzyme immunoassay of thromboxane B2

An immunoassay for thromboxane B2 was developed in which the hapten molecule was labeled with beta-galactosidase. The immunoprecipitate formed after competition between enzyme-labeled and unlabeled thromboxane B2 was subjected to a fluorometric assay of beta-galactosidase. Thromboxane B2 was detectable in the range of 0.1-30 pmol. Both enzyme immunoassay and radioimmunoassay showed essentially the same cross-reactivities with other prostaglandins and their metabolites when the same antibody was used. Known amounts of thromboxane B2 were added to human plasma, and the sample was applied to an octadecyl silica column. The extract was analyzed by enzyme immunoassay to examine the correlation between the added (x) and measured (y) thromboxane B2 (y = 1.09x + 11.07 pmol/ml, r = 0.99). A satisfactory correlation was observed between radioimmunoassay (x) and enzyme immunoassay (y) (y = 0.92x + 4.64 pmol/ml, r = 0.96). The validity of enzyme immunoassay was also confirmed by gas chromatography-mass spectrometry of a dimethylisopropylsilyl ether derivative of thromboxane B2 methyl ester. The method was applicable to the assay of thromboxane B2 produced from endogenous precursor during thrombin-induced aggregation of human platelets.     

Pulmonary microvascular response to LTB4: effects of perfusate composition

We examined the effects of leukotriene B4 (LTB4) on pulmonary hemodynamics and vascular permeability using isolated perfused guinea pig lungs and cultured monolayers of pulmonary arterial endothelial cells. In lungs perfused with Ringer solution, containing 0.5 g/100 ml albumin (R-alb), LTB4 (4 micrograms) transiently increased pulmonary arterial pressure (Ppa) and capillary pressure (Pcap). Pulmonary edema developed within 70 min after LTB4 injection despite a normal Pcap. The LTB4 metabolite, 20-COOH-LTB4 (4 micrograms), did not induce hemodynamic and lung weight changes. In lungs perfused with autologous blood hematocrit = 12 +/- 1%; protein concentration = 1.5 +/- 0.2 g/100 ml), the increases in Ppa and Pcap were greater, and both pressures remained elevated. The lung weight did not increase in blood-perfused lungs. In lungs perfused with R-alb (1.5 g/100 ml albumin) to match the blood perfusate protein concentration, LTB4 induced similar hemodynamic changes as R-alb (0.5 g/100 ml) perfusate, but the additional albumin prevented the pulmonary edema. LTB4 (10(-11)-10(-6) M) with or without the addition of neutrophils to the monolayer did not increase endothelial 125I-albumin permeability. Therefore LTB4 induces pulmonary edema when the perfusate contains a low albumin concentration, but increasing the albumin concentration or adding blood cells prevents the edema. The edema is not due to increased endothelial permeability to protein and is independent of hemodynamic alterations. Protection at higher protein-concentration may be the result of LTB4 binding to albumin.     

A laboratory study of the copper and nickel uptake and regulation in a copper-tolerant decapod, Cambarus bartoni (Fabricius) (Decapoda, Crustacea)

Tissue copper concentration in whole Cambarus bartoni, exposed for four weeks to 0.125, 0.25 and 0.5 mg Cu/litre and 0.2, 0.4 and 0.8 mg Ni/litre in the laboratory at 18 degrees C, showed a linear relationship with exposure time at 0.25 and 0.5 mg Cu/litre; the regression slope at 0.5 mg Cu/litre (r2 = 0.218; 0.001 less than P less than or equal to 0.05) was approximately two times higher than that at 0.25 mg Cu/litre (r2 = 0.148; 0.025 less than P less than or equal to 0.05). Regression analysis of nickel concentrations in whole crayfish as a function of exposure time showed a third degree polynomial relationship at 0.4 (y = 49.989 + 14.77x - 15.395x2 + 3.194x3) and 0.8 (y = 52.053 + 37.05x - 28.933x2 + 5.133x3) mg Ni/litre, as well as in the control (y = 49.126 + 27.253x - 23.468x2 + 4.168x3) (y = Ni concentration; x = exposure time in weeks), suggesting the occurrence of a Ni flux every 4 weeks.     

Continuous measurement of protein osmotic pressure in blood and lymph of sheep

We have continuously measured protein osmotic pressure of blood and lymph in sheep to compare two kinds of needle osmometers (rigid and flexible) with a membrane osmometer (Wescor). We also compared the averaged values of the continuous measurement with osmotic pressure calculated from total protein and albumin fraction, using the Yamada equation. The rigid-needle and membrane osmometers showed excellent correlation (y = 1.00x + 0.06; r greater than 0.99). The flexible-needle osmometer tended to overestimate osmotic pressure (avg 16%). We used the rigid-needle osmometer for continuous measurements of protein osmotic pressure of blood and lymph in anesthetized or unanesthetized sheep to observe changes in protein osmotic pressure of blood and lymph through the three different interventions. The relationship between the theoretical values (x) and the continuous measurements (y) of osmotic pressure was good (y = 0.99x + 0.16, r = 0.97), but after various interventions, the continuously measured protein osmotic pressure tended to exceed the calculated measurements. The continuous measurement should be monitored with spot samples measured in a stationary osmometer or by calculation of osmotic pressure from total protein concentration and albumin fraction.     

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE.     

Metabolism of rats running up and down an incline

The purpose of these experiments was to determine oxygen consumption (VO2) in rats as a function of treadmill speed (10, 20, 30, 40, and 50 m . min-1) as they ran on the level and up and down a 16 degree (17.8%) incline. The slopes of the regression lines relating VO2 (ml O2 . kg-1 . min-1) to running speed (m . min-1) were linear for all three inclines. The regression slope for uphill runners (y = 1.25x + 47.7) was greater than the regression slopes for level (y = 0.88x + 41.2) (P less than 0.025) or downhill (y = 0.68x + 39.7) (P less than 0.005) runners, and the regression slope for level runners was greater than that for downhill runners (P less than 0.10). All VO2 measurements were submaximal. In conclusion, incline has a significant effect on the metabolism of rats running on a motor-driven treadmill.     

Reference values of pulmonary diffusing capacity for nitric oxide in an adult population.

Our objective was to create reference values for single-breath DLNO based on a sample of non-smoking healthy males and females using a short breath-hold time. The sample included 130 individuals varied in age (18-85 yr), height (149-190 cm), and weight (49.4-102.6 kg). The subjects performed single-breath-hold maneuvers at rest inhaling 41 +/- 6 ppm NO and a standard diffusion mixture. The breath-hold time was 5.5 +/ -0.6 s. Multiple linear regression with backward elimination of the independent variables age, weight, gender, and either measured lung volume (called alveolar volume or VA) or height revealed specific prediction equations for DLNO. Inserting VA instead of height into the regression equation determined how much of an abnormality of DLNO was due to gas exchange versus low lung volume. The predicted DLNO adjusted for lung volume (ml/min/mmHg) = DLNO = 73.1 + 17.26 x (VA)+17.56 x (gender) - 1.0 x (age). The predicted DLNO unadjusted for lung volume (ml/min/mmHg) = -20.1 + 1.167 x (height)+31.81 x (gender) - 1.21 x (age). For gender, 1 = males, 0 = females; VA = liters; height = cm. Age, gender and VA (lung volume) were the best predictors of DLNO and DLCO. Weight was not a good independent predictor of DLNO or DLCO. When normalizing for height and age, women have 650 ml lower forced vital capacity, 660 ml lower VA, and a 6 and 32 ml/min/mmHg lower DLCO and DLNO, respectively, compared to men. Normalizing for lung volume and age, women have, on average, a 3.2 and 18 ml/min/mmHg lower DLCO and DLNO, respectively, compared to men.     

Automated, fast and sensitive quantification of 17 alpha-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction

Plasma 17 alpha-hydroxyprogesterone (17-OHP), androstenedione and testosterone measurements are important for the diagnosis and monitoring of hyperandrogenic disorders, most importantly for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. The reliability of immunoassays has proved questionable especially for newborns and children. In order to reduce the analytical interferences due to cross-reactivity or matrix effects, to improve accuracy and shorten the analysis time, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with atmospheric pressure chemical ionization (APCI) for simultaneous measurement. An on-line extraction cartridge with column-switching technique and liquid chromatography over a Chromolith RP 18 e column allow a rapid and easy quantification. The lowest limit of detection was 0.03-0.06 microg/L. Our method has proved linear up to 250 microg/L (r=0.999). Recoveries (S.D.) of 17-OHP, androstenedione and testosterone in plasma were 100% (5), 102% (2) and 92% (4), respectively. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for 17-OHP (excluding neonate samples) was y=1.942 x+0.255 nmol/L (r=0.695; n=97). In comparison to our values, the immunoassay generally overestimates steroid concentration. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for testosterone was y=0.963 x+0.035 nmol/L (r=0.955; n=107). Preliminary reference intervals for children were determined as a function of age and sex. The sensitivity and specificity of the LC-MS/MS method offer advantages over routine immunoassays due to the elimination of interferences especially for newborns, high throughput and short chromatographic run time.     

Onset of secretion of proteins with antiviral activity by pig conceptuses

In Exp. 1, antiviral activity was detected in Day-15 pregnant uterine flushings (6222 +/- 2167 units/ml) and in conceptus culture medium collected at 0, 1, 2, 4, 8, 16, 24, 32, 40, and 48 h (95, 375, 650, 1216, 1600, 2100, 2017, 2083, 3500 and 5000 units/ml, respectively; R2 = 0.81, P less than 0.01; y = 190.0 + 252.7x - 11.2x2 + 0.2x3. In Exp. 2, antiviral activity of Day-15 conceptus culture medium was reduced 99% after boiling for 20 min (P less than 0.01) and, after 18 h dialysis (6000-8000 Mr cut-off), 100% of the activity was in the retentate. In Exp. 3, antiviral activity was not detected in cultures of conceptuses from Days 10 and 11 and activity was maximal for Day 14 and Day 15 conceptuses (2100 and 2083 units/ml, respectively). Effects of day were best described by a quadratic regression equation (y = 17,652 - 3263x + 150x2; R2 = 0.55, P less than 0.01). In Exp. 4, changes in antiviral activity detected in uterine flushings from pregnant gilts on Days 8, 10, 11, 12, 14 and 15 (1.3, 0, 6.7, 63.3, 580 and 1663 units/ml, respectively) were described by the equation y = -20,743 + 6189x - 606x2 + 20x3 (R2 = 0.85, P less than 0.01). In Exp. 5, low antiviral activities (5-30 units/ml) were detected in all plasma samples collected from the uterine artery and uterine vein of pregnant and cyclic gilts, but values were not significantly influenced by pregnancy status, day or site of collection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Method of determining proteolytic activity by using a conjugate of bovine serum albumin with peroxidase

The authors propose a method for determination of proteolytic activity, based on the hydrolysis of peroxidase-labeled molecules of bovine serum albumin immobilized on the surface of polystyrene microassay plates with the subsequent determination of peroxidase activity on the carrier or in the solution. The optimum conditions for the sorption of the labeled substrate have been established. The method permits the determination of bacillary alkaline protease at a concentration of 01. microgram/ml within 45 minutes. The determination of four proteases has demonstrated that this method shows good correlation with the routine one (r = 0.98), but is more sensitive and less time- and labor-consuming.     

Determination of concentrations of flecainide in human serum by high-performance liquid chromatography on a fluorocarbon-bonded silica gel column

An optimized method for the determination of flecainide in serum is presented. Extraction using a solid-phase C₁₈ column and chromatography on a stabilized fluorocarbon-bonded silica gel column effectively separate flecainide from an internal standard (a positional isomer of flecainide). The HPLC apparatus and conditions were as follows: analytical column, Fluofix 120N; sample solvent, 20 μl; column temperature, 40°C; detector, Shimadzu RF-5000 fluorescence spectrophotometer (excitation wavelength=300 nm, emission wavelength=370 nm); mobile phase, 0.06% phosphoric acid containing 0.1% tetra-n-butyl ammonium bromide–acetonitrile (75:25, v/v); flow-rate, 1.0 ml/min. The standard curves for flecainide were linear in the concentration range examined (10–2000 ng/ml). The regression equation was y=0.08+0.0078x (r=0.9998). The minimum detectable amount of flecainide was approximately 5 ng/ml. In the within-day study, the precision coefficients of variation were 2.66, 2.18, 2.54, 2.72, 2.88, 2.24, and 3.29% for the 10, 50, 100, 200, 500, 1000, and 1500 ng/ml standards, respectively. The absolute recovery rates of flecainide at each concentrations were 94–100%. The method described provides analytical sensitivity, specificity and reproducibility suitable for both biomedical research and therapeutic drug monitoring.     

Effect of hemorrhage on hepatic tissue blood flow in the domestic fowl (Gallus domesticus)

1. Thermal Pulse Decay (TPD) methodology was used to monitor hepatic tissue blood flow (hepatic perfusion) in anesthetized birds prior to and during hemorrhagic hypotension. 2. Hemorrhage was accomplished by periodic removal of blood through a carotid cannula. Reducing the estimated blood volume (BV) from 100 to less than 50% decreased hepatic perfusion from 4.36 +/- 0.7 to 1.88 +/- 0.7 ml/min/g. 3. Changes in hepatic perfusion during the experiment were related to mean arterial blood pressure (MABP) by the following linear regression equation: hepatic perfusion = -1.79 +/- 0.0807x (r2 = 0.57).