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Objective: To compare the accuracy of a 3rd (Dolphin Voyager) versus 1st generation pulse oximeter (Nellcor N-180)., Study Design: Prospective laboratory investigation., Animals: Eight adult dogs., Methods: In anesthetized dogs, arterial oxygen saturation (SpO(2)) was recorded simultaneously with each pulse oximeter. The oxygen fraction in inspired gas (FiO(2)) was successively reduced from 1.00 to 0.09, with re-saturation (FiO(2) 0.40) after each breathe-down step. After each 3-minute FiO(2) plateau, SpO(2) and pulse rate (PR) were compared with the fractional arterial saturation (SaO(2)) and PR determined by co-oximetry and invasive blood pressure monitoring, respectively. Data analysis included Bland-Altman (B-A) plots, Lin's concordance correlation factor (rho(c)), and linear regression models., Results: Over a SaO(2) range of 33-99%, the overall bias (mean SpO(2) - SaO(2)), precision (SD of bias), and accuracy (A(rms)) for the Dolphin Voyager and Nellcor N-180 were 4.3%, 4.4%, and 6.1%, and 3.2%, 3.0%, and 4.3%, respectively. Bias increased at SaO(2) < 90%, more so with the Dolphin Voyager (from 1.6% to 8.6%) than Nellcor N-180 (from 3.2% to 4.5%). The SpO(2) readings correlated significantly with SaO(2) for both the Dolphin Voyager (rho(c) = 0.94) and Nellcor N-180 (rho(c) = 0.97) (p < 0.001). Regarding PR, bias, precision, and accuracy (A(rms)) for the Dolphin Voyager and Nellcor N-180 were -0.5, 4.6, and 4.6 and 1.38, 4.3, and 4.5 beats minute(-1), respectively. Significant correlation existed between pulse oximeter and directly measured PR (Dolphin Voyager: rho(c) = 0.98; Nellcor N-180: rho(c) = 0.99) (p < 0.001)., Conclusions and Clinical Relevance: In anesthetized dogs with adequate hemodynamic function, both instruments record SaO(2) relatively accurately over a wide range of normal saturation values. However, there is an increasing overestimation at SaO(2) < 90%, particularly with the Dolphin Voyager, indicating that 3rd generation pulse oximeters may not perform better than older instruments. The 5.4-fold increase in bias with the Dolphin Voyager at SaO(2) < 90% stresses the importance of a 93-94% SpO(2) threshold to ensure an arterial saturation of >or=90%. In contrast, PR monitoring with both devices is very reliable.

Systemic function and oxygenation changes during hemorrhagic shock treatment were continuously monitored and correlated with real-time microvascular changes. After splenectomy, each dog (n = 12) was hemorrhaged (MAP = approximately 50 mmHg; approximately 40% blood loss = 32-36 ml/kg) and randomly assigned to 4 resuscitation groups: autologous/shed blood, hemoglobin-based oxygen-carrier/Oxyglobin, crystalloid/saline, and colloid/Hespan. Systemic function and oxygenation changes were continuously monitored and measured using standard operating room protocols. Computer-assisted intravital microscopy was used to non-invasively videotape and objectively analyze and quantify real-time microvascular changes in the conjunctival microcirculation. All measurements were made during pre-hemorrhagic (baseline), post-hemorrhagic and post-resuscitation phases of the study. Pre-hemorrhagic microvascular changes were similar in all 12 dogs (venular diameter = 43 +/- 12 microm; red-cell velocity = 0.6 +/- 0.2 mm/s). All dogs showed similar significant (P<0.01) post-hemorrhagic microvascular changes: approximately 20% decrease in venular diameter; approximately 80% increase in red-cell velocity. These microvascular changes correlated with post-hemorrhagic systemic function and oxygenation changes. The resuscitations restored microvascular changes to pre-hemorrhagic values; the microvascular reversals also correlated with post-resuscitation systemic function changes in all groups. However, only shed blood resuscitation restored oxygenation level close to pre-hemorrhagic values. All 12 dogs survived resuscitation treatments despite differences in oxygen-carrying capability between groups.

Hemoglobin-glutamer-200 (HBOC-200) is a hemoglobin (Hb)-based oxygen carrier (HBOC) comprising glutaraldehyde-polymerized bovine Hb. In this study, we sought to determine the pharmacokinetics of this first generation HBOC after IV infusion of 32.5 g of HBOC-200 solution in horses. Quantification of HBOC-200 in equine plasma and urine was performed using a method recently developed by our laboratory. The elimination from plasma was based on size distribution of the bovine Hb polymer. The decline of plasma concentration-time curve of HBOC-200 was described by a noninterchanging 2-compartmental model. The median elimination half-lives of the small and large aggregates were 1.3 and 12.0 h, respectively. Of the HBOC-200 infused, 47.0% was eliminated as the smaller molecular weight and 53% as the larger molecular weight polymers. The area under the plasma concentration-time curve was 5143.1 microg.h(-1).mL(-1). The volumes of distribution of the small and large aggregates were 86.9 and 63.9 mL/kg and the clearances were 42.1 and 3.8 mL.kg(-1).h(-1), respectively. In conclusion, elimination of first generation HBOCs was shown to be more complex than previously assumed because of the heterogeneous nature of these solutions. Mammalian species dispose of Hb using similar mechanisms, and there is no unique metabolic process in the horse that would not allow a logical extension of the general interpretation of this study.

The superficial (SDF) and deep digital flexor (DDF) muscles are critical for equine forelimb locomotion. Knowledge of their mechanical properties will enhance our understanding of limb biomechanics. Muscle contractile properties derived from architectural-based algorithms may overestimate real forces and underestimate shortening capacity because of simplistic assumptions regarding muscle architecture. Therefore, passive and active (=total - passive) force-length properties of the SDF and DDF muscles were measured directly in vivo. Muscles from the right forelimbs of four Thoroughbred horses were evaluated during general anesthesia. Limbs were fixed to an external frame with the muscle attached to a linear actuator and load cell. Each muscle was stretched from an unloaded state to a range of prefixed lengths, then stimulated while held at that length. The total force did not exceed 4000 N, the limit for the clamping device. The SDF and DDF muscles produced 716+/-192 and 1577+/-203 N maximum active isometric force (F(max)), had ascending force-length ranges (R(asc)) of 5.1+/-0.2 and 9.1+/-0.4 cm, and had passive stiffnesses of 1186+/-104 and 1132+/-51 N/cm, respectively. The values measured for F(max) were much smaller than predicted based on conservative estimates of muscle specific tension and muscle physiological cross-sectional area. R(asc) were much larger than predicted based on muscle fiber length estimates. These data suggest that accurate prediction of the active mechanical behavior of architecturally complex muscles such as the equine DDF and SDF requires more sophisticated algorithms.

We sought to determine whether hemoglobin-based oxygen carriers (HBOCs), hemoglobin glutamer-200 [bovine] (HBOC-200, Oxyglobin), hemoglobin glutamer-250 [bovine] (HBOC-201, Hemopure), and hemoglobin raffimer (Hemolink) interfere with the accuracy of lactate measurements. Combinations of concentrated L-lactate solution, HBOC, and blood or plasma with added PlasmaLyte-A were added to sample tubes to make a linear and constant increase in lactate concentration in consecutive samples. Sample lactate concentrations ranged from 5-110 mg/dL (0.6-12 mm) (physiological reference range: 5-20 mg/dL [0.56-2.2 mm]). Comparisons were made between machine measured lactate concentrations and calculated lactate concentrations. For Hb glutamer-250, the average difference between measured and calculated lactate concentrations was -5.1 mg/dL (-0.57 mm) (LX-20), with greater underestimation at larger lactate concentrations. For Hb raffimer, the average difference was -2.2 mg/dL (-0.24 mm) (LX-20). The veterinary product, Hb glutamer-200, was tested on 3 analyzers (LX-20(R), YSI 1500, and YSI 2300). The YSI 1500 was the most accurate instrument with the mean difference between measured minus calculated lactate being +1.3 mg/dL versus -2.6 mg/dL (YSI 2300) and -8.4 mg/dL (LX-20). The clinical implications of this study are that with increasing levels of an HBOC in plasma, lactate interpretation may become inaccurate, especially at larger lactate concentrations, causing underestimation of measured lactate values and possible under-treatment of the patient. Therefore, caution must be exercised when interpreting lactate results when a HBOC is present in plasma.

In this study, we validated the accuracy of lactate measurements (YSI 2700 SELECT glucose/lactate analyzer) in the presence of methemoglobin from an oxidized bag of hemoglobin-based oxygen carrier (Met-HBOC), hemoglobin glutamer-200 (Oxyglobin; Biopure Corp). Different combinations of concentrated L-lactate solution, pooled canine plasma, and Plasmalyte A were added to 4 sample groups (1%, 10%, 20%, and 40% Met-HBOC [1.3 g/dL]) to yield linear increases in lactate concentration in consecutive samples. The mean difference between measured and calculated lactate was -5.1 mg/dL (1% Met-HBOC), -5.8 mg/dL (10% Met-HBOC), -4.6 mg (20% Met-HBOC), and -8.5 mg/dL (40% Met-HBOC). The root mean square error was 6.5 mg/dL, 7.4 mg/dL, 6.8 mg/dL, and 10.3 mg/dL, respectively. The Bland-Altman correlation (r) was r = -0.94 (P = 0.01), r = -0.91 (P < 0.001), r = -0.90 (P < 0.001), and r = -0.94 (P < 0.001), respectively, where r = 0 for perfect agreement between measured and calculated values. Results indicate that true lactate levels in the presence of Met-HBOC are underestimated when measured by an YSI 2700 analyzer independent of the amount of Met-HBOC present. When interpreting lactate concentrations from a patient with a HBOC present in plasma, underestimation of true lactate levels may occur unrelated to methemoglobin concentrations.

Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human plasma. OXY, HMP, and HML were extracted from equine or human plasma by solid-phase extraction using Bond Elut ENV cartridges and were digested by trypsin at 37 degrees C for 3 h. The tryptic digests were analyzed by LC-MS/MS, and tryptic peptides specific for bovine and human Hbs were targeted. OXY and HMP were detected, quantified, and confirmed using the y14 ion and b8 ion of the tryptic peptide from bovine Hb alpha chain residues 69-90, and HML was quantified using the tryptic peptide from human Hb alpha chain residues 63-91. The limit of detection for OXY in equine plasma and HML in human and equine plasma was 50 and 250 microg/mL for HMP in human and equine plasma. The limit of confirmation was 250 microg/mL for OXY in equine plasma, 500 microg/mL for HML in human and equine plasma, and 1000 microg/mL for HMP in human and equine plasma. The linear range for quantification was 50-5000 microg/mL for OXY in equine plasma and for HML in human and equine plasma, and 250-5000 microg/mL for HMP in human and equine plasma. The intraday and interday CV were less than 17% for quantification of OXY in equine plasma with external calibration. OXY was stable for more than 30 days at -20 and -70 degrees C. OXY was detected and quantified in equine plasma up to 24 h following administration of a very low dose of OXY (32.5 g in 2 x 125 mL per horse), and its presence in equine plasma was confirmed up to 12 h postadministration., (Copyright 2004 American Chemical Society)

Hemoglobin-based oxygen carriers (HBOCs) of bovine hemoglobin (Hb) or human Hb origin were developed for replacement or augmentation of blood during transfusion and have the potential to increase oxygen-carrying capacity of circulating blood and thus improve tissue oxygen delivery. Due to their potential for increasing oxygen-carrying capacity of circulating blood, they are excellent candidates for abuse in human and equine athletes. To deter athletes from blood doping with HBOCs such as Hemopure and Oxyglobin (OXY), a method for detection, confirmation, quantification, and distinguishing of HBOCs from native hemoglobin in test samples is needed. The purpose of this study was to identify unique peptides specific for bovine Hb and human Hb that are useful in the detection and confirmation of HBOCs in test samples. The LC-MS chromatographic peak profiles of tryptic digests from OXY, bovine Hb, human Hb, and equine Hb were compared, and unique tryptic peptides specific for bovine Hb, human Hb, and equine Hb were identified. The peptides specific for bovine Hb and OXY are related to bovine Hb alpha chain residues 69-90 and beta chain residues 40-58. The peptides specific for human Hb are related to human Hb alpha chain residues 63-91 and beta chain residues 42-60 and 68-83. The amino acid sequences of these unique tryptic peptides were confirmed by their characteristic MS/MS spectra. MS/MS spectra, b-ion series and y-ion series, and LC retention time of the tryptic peptides are essential pieces of information for the unequivocal identification, detection, and confirmation of HBOCs. The results of this study provide useful and defensible data on identification, detection, and confirmation of HBOCs of bovine Hb or human Hb origin. In addition, in-ESI-source fragmentation of tryptic peptides was observed in this study. The fragmentation was undesired since it decreased intensities of the trypic peptide ions, but it was helpful to elucidating sequences of the tryptic peptides thanks to the fragment peptide ions produced from the fragmentation., (Copyright 2004 American Chemical Society)

Economic losses related to increased stress during the transport of pigs are well documented. The effects of supplementing of tryptophan (Trp), vitamin E, or a herbal product via feed or drinking water were investigated in terms of effects on stress response in pigs during transport simulation. The study consisted of three analogous experiments. For the testing in each experiment, the pigs (23.5+/-3.2 kg) were allocated to one of two treatments, with and without supplementation of a product. The applied doses were Trp (5 g/L drinking water for 3 d), vitamin E (additional amount of 300 mg/kg feed for 21 d, as-fed basis), and Sedafit (2.5 g/L drinking water for 2 d). Sedafit is a commercial herbal product containing Valeriana officinalis L. and Passiflora incarnata L. as active components. In each experiment of the study, at least 47 pigs were involved, which were treated in groups of 3. The day before transport simulation, a Holter device was attached to the pigs to produce an electrocardiogram during the night (rest values), as well as during vibration in the transport simulator (1.2 Hz, 1 m/s2), where the behavior of the pigs (standing-sitting-lying) was also observed. Samples of saliva (taken before, during, and after [3x] vibration) and blood (taken before and after vibration) were analyzed for cortisol and intermediate metabolites (glucose, lactate, creatine kinase, and nonesterified fatty acids), respectively. Pigs supplemented with Trp tended to spend more time lying down during the second hour of vibration (P < 0.05). Vitamin E decreased the peak heart rate (P < 0.05), ventricular ectopic beats (P < 0.01), and ST elevation (P < 0.10). The supplementation of Sedafit resulted in smaller increases of the investigated heart variables (minimum heart rate, P < 0.05; ventricular ectopic beats, P < 0.05; ST elevation, P < 0.01) during and after stress evocation compared with the control group. None of the tested products influenced the intermediate metabolites; one possible explanation for this finding may be that peak values were reached before the time of bleeding. In conclusion, Trp had a positive behavioral effect in this experiment, and vitamin E and Sedafit mediated an increase in some heart variables, suggesting sedative and antianxiety effects.

Blood substitute resuscitation as a treatment modality for moderate hypovolemia (approximately 40% blood loss) in a canine model has been evaluated using Oxyglobin (Biopure Hemoglobin Glutamer-200/ Bovine; a hemoglobin-based oxygen-carrier) and Hespan (6% hetastarch; a nonoxygen-carrier) as resuscitants. Autologous (shed) blood served as control. Nine dogs were studied--after splenectomy, each dog was hemorrhaged (32-36 mL/kg; MAP = approximately 50 mmHg) and randomly assigned to the three resuscitation groups. Microvascular, systemic function and oxygenation characteristics were monitored and/or measured simultaneously in prehemorrhagic (baseline), posthemorrhagic and postresuscitation phases for correlation-real-time microvascular changes in the bulbar conjunctiva were noninvasively measured via computer-assisted intravital microscopy and systemic function and oxygenation changes were monitored and/or measured via instrumentation and devices incorporated into our bioengineering station in an operating room setting. Blood chemistry was also studied for relevant measurements. Prehemorrhagic microvascular characteristics were similar in all animals (venular diameter = 41 +/- 12 microm, A:V ratio = approximately 1:2, red-cell velocity = 0.5 +/- 0.3 mm/s). All animals also showed similar prehemorrhagic systemic function and oxygenation measurements comparable to a previous study and were consistent with normal measurements in dogs. At the completion of hemorrhaging to achieve moderate hypovolemia (approximately 40% blood loss with MAP at approximately 50 mmHg), all nine animals showed similar significant (P < 0.01) posthemorrhagic microvascular changes, including approximately 17% decrease in diameter (34 +/- 7 microm), A:V ratio = variable, and approximately 80% increase in velocity (0.9 +/- 0.5 mm/s). All animals also showed similar significant (P < 0.01) posthemorrhagic systemic function and oxygenation changes, with decreases in Hct, aHb(total), MPAP, MAP, SAP, DAP, CO, SVI, CaO2, and CvO2 and increases in HR and lactic acidosis. Shed blood (control) resuscitation restored posthemorrhagic microvascular changes close to prehemorrhagic values (diameter = 39 +/- 6 microm, A:V ratio = approximately 1:2, velocity = 0.6 +/- 0.4 mm/s). Oxyglobin and Hespan restored microvascular changes in similar manner close to prehemorrhagic values (Oxyglobin: diameter = 38 +/- 3 microm, A:V ratio = approximately 1:2, velocity = 0.6 +/- 0.4 mm/s; Hespan: diameter = 38 +/- 7 microm, A:V ratio = 1:2, velocity = 0.5 +/- 0.4 mm/s). After resuscitation, shed blood (control) restored all systemic function and oxygenation changes close to prehemorrhagic values. However, both Oxyglobin and Hespan resuscitation restored systemic function changes, but not oxygenation changes, to prehemorrhagic values. This was an interesting finding because of the different oxygen-carrying capability of Oxyglobin (oxygen-carrying) and Hespan (nonoxygen-carrying). The result suggests that either volume replenishment alone (and not oxygen-carrying capability) is needed to treat moderate hypovolemia or oxygenation measurements obtained by standard methods (oximetry, blood chemistry) may not reflect tissue oxygenation levels.

Stevioside orally administered to pigs was completely converted into steviol by the bacteria of the colon. However, no stevioside or steviol could be detected in the blood of the animals, even not after converting steviol into the (7-methoxycoumarin-4-yl)methyl ester of steviol, a very sensitive fluorescent derivative with a detection limit of about 50 pg. The intestinal transport characteristics of stevioside, rebaudioside A and steviol were also studied in the Caco-2 system. Only a minor fraction of stevioside and rebaudioside A was transported through the Caco-2 cell layer giving a Papp value of 0.16x10(-6) and 0.11x10(-6) cm/s, respectively. The Papp value for the absorptive transport of steviol was about 38.6x10(-6) cm/s while the Papp value for the secretory transport of steviol was only about 5.32x10(-6) cm/s suggesting carrier-mediated transport. The discrepancy between the relatively high absorptive transport of steviol and the lack of steviol in the blood may be explained by the fact that in the Caco-2 study, steviol is applied as a solution facilitating the uptake, whereas in the colon steviol probably is adsorbed to the compounds present in the colon of which the contents is being concentrated by withdrawal of water.

Objective: To develop and test an experimental model for in vivo short-term recording of peak isometric forces of the digital flexor muscles in the forelimb of adult horses., Study Design: In vivo experimental study., Sample Population: Four healthy, anesthetized, adult Thoroughbred horses (3 to 7 years old; 527 +/- 87 kg), Methods: In dorsal recumbency, ulnar and median nerves were exposed and instrumented with insulated bipolar cuff stimulation electrodes for later connection to an electrical stimulator. In left lateral recumbency, a biplanar fixator was applied to the right humerus and a custom-made, rigid, aluminum frame connected to it, to allow loading of muscles distal to the fixator. Threaded transfixation pins through the radial and metacarpal condyles were clamped to the rigid frame so that the humerus, radius, ulna, and metacarpus were fixed in position. Each digital flexor muscle insertion tendon was transected just above the metacarpophalangeal joint, extracted from the carpal canal, and secured in a metal clamp positioned at the distal myotendinous (MT) junction. Distally, the clamp was connected in series to a load cell and a pneumatic actuator to record force and to maintain muscle length during nerve stimulation. A linear potentiometer was connected in parallel to the actuator to record MT junction position. Initial trials were conducted to identify median and ulnar nerve stimulation variables to achieve maximal muscle contraction. Isometric contractions were performed at different muscle lengths and peak forces registered during 3 seconds of supramaximal dual (ulnar and median) nerve stimulation., Results: A stimulation voltage of 2.5 to 5.0 V at 50 Hz usually produced maximal force for both the superficial digital flexor (SDF) and deep digital flexor (DDF) muscles. Single ulnar and median nerve stimulation elicited force development not only in the DDF muscle but also in the SDF muscle. At voltages higher than 1 V, normalized force was greatest with combined median and ulnar nerve stimulation for both the DDF and SDF muscles; however, normalized force was greater for median nerve stimulation than ulnar nerve stimulation in the DDF muscle, and the opposite relationship was observed for the SDF muscle. Final recording of dual supramaximal nerve stimulation of SDF and DDF muscles resulted in peak isometric forces of 716 +/- 192 N and 1,577 +/- 203 N, respectively., Conclusions: The instrumentation technique and experimental protocol enabled recording of peak isometric forces in the SDF and DDF muscles of anesthetized adult horses., Clinical Relevance: Studies using this model will improve knowledge of SDF and DDF muscle mechanics with insight to functional implications of the complex architecture of these muscles. Knowledge of the dynamic performance of the SDF and DDF muscles would also be useful for the development of new treatment strategies for flexor deformities and tendon injuries in horses., (Copyright 2003 by The American College of Veterinary Surgeons)

Unlabelled: We measured lead concentrations in three hemoglobin-based oxygen carriers (HBOCs; Oxyglobin, Hemopure, and Hemolink) and compared them with lead concentrations from blood-bank blood. Oxyhemoglobin dissociation was measured with large concentrations of lead in bovine HBOC, with or without bovine blood, and in bovine blood. Samples of each were prepared by combining one with normal saline (control), the second with small lead concentrations (22 micro g/dL), and the third with toxic lead concentrations (70 micro g/dL). They were blended in 2 tonometers at oxygen concentrations (2.5%, 5%, 8%, 10%, 21%, and 95%) with 5% CO(2) and the remainder nitrogen for 5 min per sample after a 15-min wash-in with each level of oxygen and were measured with co-oximetry. Oxygen saturation was plotted against PO(2), fitting fourth-order polynomial nonlinear regression to the data. The lead concentrations of the three HBOCs were 0.51, 0.22, 0.40 micro g/dL. There were no clinically important differences of the oxyhemoglobin dissociation curves as a function of lead concentration. The lead concentrations of the three tested HBOCs were small and no larger than the average for blood-bank blood. The presence of increasing concentrations of lead in either concentrated solution of bovine HBOC or a 1:1 mixture of bovine HBOC and native bovine blood does not appear to affect hemoglobin oxygenation in an acute in vitro model of increased lead concentrations., Implications: Gunshot wounds rapidly increase circulating lead concentrations. Lead concentrations are small in three hemoglobin-based oxygen carriers (HBOCs), and HBOCs and/or bovine blood do not appear to be affected by lead concentrations in terms of immediate oxygen on-loading and off-loading. HBOCs may be useful in patients with gunshot wounds.

Objective: To compare effects of 6% hetastarch (Hextend) and hemoglobin-based oxygen carrier hemoglobin glutamer-200 (Hb-200) (bovine; Oxyglobin) on hemodynamics, arterial oxygen content, and systemic oxygen delivery in a canine hemorrhagic shock model., Design: Randomized laboratory investigation., Setting: University surgical research facility., Subjects: Twenty-four anesthetized healthy, adult, mongrel dogs (28 +/- 1 kg; 7 female, 17 male)., Interventions: Dogs were instrumented for determinations of heart rate, arterial, central venous, pulmonary arterial, and pulmonary arterial occlusion pressures, and cardiac index. Total solids, colloid oncotic pressure, arterial oxygen content, Hb, lactate, pH, and blood gases were analyzed in blood samples. Recordings were made before, after 1 hr of hemorrhagic shock, and immediately and 3 hrs after infusion of either 30 mL/kg hetastarch (group 1), 10 mL/kg Hb-200 + 20 mL/kg hetastarch (group 2), 20 mL/kg Hb-200 + 10 mL/kg hetastarch (group 3), or 30 mL/kg Hb-200 (group 4)., Measurements and Main Results: Hemorrhage (35 +/- 1 mL/kg) reduced mean arterial pressure to 50 mm Hg and caused significant decreases in total Hb, mean pulmonary arterial pressure, cardiac index and systemic oxygen delivery, increases in heart rate and systemic vascular resistance, and lactic acidosis. In group 1, hetastarch infusion was accompanied by increases of pulmonary arterial pressure, cardiac index, and blood oxygen extraction above baseline, and decreases of systemic vascular resistance, total Hb, total solids, arterial oxygen content, and systemic oxygen delivery below baseline (p <.05). Other data returned to baseline. In groups 2 to 4, hemodynamic functions (except pulmonary arterial pressure) recovered, yet neither total Hb (i.e., plasma and red blood cell Hb) nor arterial oxygen content increased despite increases in plasma Hb of 2 to 5 g/dL and proportionate increases in total solids. Systemic oxygen delivery improved dose-dependently with Hb-200 but did not return to baseline (p <.05), reaching values comparable to hetastarch group only at 30 mL/kg Hb-200. In all groups, oxygen extraction remained above baseline. Metabolic acidosis and lactatemia resolved significantly faster in groups 2 to 4, and colloid oncotic pressure after resuscitation was greater in groups 2 to 4 than in controls (p <.05)., Conclusions: In hemorrhagic shock, Hb-200 infusion may not improve oxygen delivery more than hetastarch, likely due to hemodilution caused by its high colloid oncotic pressure, but may facilitate diffusive oxygen transport to tissues.

Unlabelled: In this study, we evaluated the validity of saturation measurements in mixed venous and arterial blood during posthemorrhagic anemia and resuscitation with varying levels of hemoglobin-based oxygen carrier (Hemoglobin glutamer-200 [bovine]; Oxyglobin [Hb-200]). Nineteen anesthetized, splenectomized, mixed-breed dogs were anesthetized (two were excluded from the data because they did not survive the exsanguination, supporting the validity of the model). Their pulmonary arteries were cannulated with the Abbott QVUE Oximetrix 3 catheter. An 18-gauge catheter was placed in the femoral artery, and a reusable Nellcor probe was applied to the tongue. Mixed venous and arterial samples were drawn at baseline, after 40% hemorrhage (to keep arterial pressure at 50 mm Hg), and postresuscitation with 30 mL/kg of 6% hetastarch in lactated Ringer's solution (n = 4), 10 mL/kg of Hb-200, 20 mL/kg of hetastarch (n = 6), 20 mL/kg of Hb-200, and 10 mL/kg of hetastarch (n = 7). Samples were compared with oxygen content from the LEXO2CON-K oxygen analyzer, and oxygen content was calculated for all values from the monitors. Results were compared by using analysis of variance. There was good correlation (0.97 > or = r > or = 0.92) for the measured versus calculated hemoglobin oxygen saturation values at baseline. After resuscitation, the correlation between calculated and measured values of oxygen content was significantly smaller for all tested instruments. The values of oxygen content calculated from the oxygen saturation monitor and from the oximetric pulmonary artery can deviate by as much as 20% from directly measured values. We conclude that the administration of this oxygen therapeutic may interfere with the values of some monitors., Implications: This study evaluated oxygen saturation monitors in a canine model of acute blood loss and resuscitation with a blood substitute and found that these may interfere with the monitors' results in a dose-dependent way.

This study was designed to validate oxygen saturation measurements from the NOVA CO-Oximeter (NOVA Biomedical Corporation, Waltham, MA), the i-STAT System (Sensor Devices, Waukesha, WI), and the Corning 170 blood gas analyzer (Bayer Corporation, East Walpole, MA) under conditions similar to the clinical application of a hemoglobin-based oxygen carrier (HBOC, hemoglobin glutamer-200 [bovine]; Oxyglobin, Biopure Corporation, Cambridge, MA). A canine model was used for both in vitro and in vivo experiments. In vivo experiments were conducted in a canine laboratory, and in vitro experiments were conducted in a tonometry laboratory. Study subjects were six mixed-breed dogs, each weighing approximately 30 kg. In the first set of experiments, the target blood po(2) levels were reached by tonometry. In the second set of experiments, quantitative measurements of total oxygen content with the LEXO2CON-K (HOSPEX Fiberoptics, Chestnut Hill, MA) were performed, immediately followed by measurements with the NOVA CO-Oximeter and the i-STAT system. HBOC was added in concentrations of 16.2, 32.5, 65, and 97.5 g/L. To analyze the clinical significance of the differences in the results obtained with the each investigated instrument, blood samples from dogs treated with HBOC after acute hemorrhagic shock were used. Oxygen saturation, oxygen content, and po(2) were measured. There was a strong correlation between the oxygen saturation values measured with the investigated instruments in samples after tonometry and known po(2). The total calculated oxygen content varied by 5% based on results generated by calculations using the investigated instruments. The results did not change with different oxygenation of the sample. The differences among methods were not significant when the HBOC concentration was 16.2 g/L. Higher concentrations of HBOC increased the difference between calculated and measured oxygen content; the i-STAT system demonstrated a greater deviation compared with the results of the other two instruments. Systemic oxygen uptake using the investigated instruments showed a high correlation with values based on LEXO2CON-K measurements (R = 0.97 for CO-Oximeter, R = 0.96 for Corning 170 blood gas analyzer, and R = 0.79 for i-STAT system). Systemic oxygen uptake values based on CO-Oximeter and Corning 170 blood gas analyzer data showed 75% accuracy; i-STAT system accuracy was 63% for control samples and 50% for samples after HBOC infusion.

Unlabelled: The accuracy of the HemoCue Plasma/Low Hemoglobin System was validated in vitr. with low levels of hemoglobin-based oxygen carriers (HBOCs). Repeated measurements were performed on 50 samples of canine plasma, each mixed with three different HBOCs at varying small concentrations (a total of 150 samples), by using plasma samples without HBOCs as controls. Two technicians performed the measurements and randomly tested each sample 10 times. The results were analyzed for correlation, and analysis of variance was used to evaluate statistical significance, with a P value of

The volatile anaesthetic sevoflurane is degraded to fluoride (F-) and a vinyl ether (Compound A), which have the potential to harm kidney and liver. Whether renal and hepatic injuries can occur in horses is unknown. Cardiopulmonary, biochemical and histopathological changes were studied in six healthy thoroughbred horses undergoing 18 h of low-flow sevoflurane anaesthesia. Serum F- concentrations were measured and clinical laboratory tests performed to assess hepatic and renal function before and during anaesthesia. Necropsy specimens of kidney and liver were harvested for microscopic examination and compared to pre-experimental needle biopsies. Cardiopulmonary parameters were maintained at clinically acceptable levels throughout anaesthesia. Immediately after initiation of sevoflurane inhalation, serum F- levels began to rise, reaching an ongoing 38-45 micromol 1(-1) plateau at 8 h of anaesthesia. Serum biochemical analysis revealed only mild increases in glucose and creatinine kinase and a decrease in total calcium. Beyond 10 h of anaesthesia mild, time-related changes in urine included increased volume, glucosuria and enzymuria. Histological examination revealed mild microscopic changes in the kidney involving mainly the distal tubule, but no remarkable alterations in liver tissue. These results indicate that horses can be maintained in a systemically healthy state during unusually prolonged sevoflurane anaesthesia with minimal risk of hepatocellular damage from this anaesthetic. Furthermore, changes in renal function and morphology observed after sevoflurane inhalation are judged minimal and appear to be clinically irrelevant; they may be the result of anaesthetic duration, physiological stressors, sevoflurane (or its degradation products) or other unkown factors associated with these animals and study conditions.

The aim of this study was to investigate the changes in circulating blood volume (CBV) after infusions of Hemoglobin glutamer-200 (bovine; Hb-200) in combination with 6% hetastarch in an animal model of acute posthemorrhagic anemia. Fifteen rabbits with a mean body weight of 2.2 +/- 0.2 kg were studied. After determination of the circulating plasma volume (CPV) and CBV, one third of the CBV was withdrawn via an arterial catheter. An equal volume of Hb-200 was then infused in the 5 animals in study group 1. In the remaining 10 animals, the same volume of 6% hetastarch solution was infused. Fifteen minutes after completion of the infusion, an additional infusion of 6% hetastarch at a dose equal to one third of the baseline CBV was performed in study group 1 and the control group. In study group 2, equal volumes of Hb-200 were infused. CPV measurements were performed at baseline, after hemorrhage, 5 and 15 minutes after volume replacement, and 1 and 15 minutes after hypervolemic hemodilution using the Evans blue dye dilution technique. The mean baseline CPV value was 53.4 +/- 1.1 mL/kg. The mean CBV at baseline was 90.8 +/- 4.9 mL/kg. After one third of the CBV was withdrawn, no differences in CBV or CPV were observed between the three groups. Replacement of one third of the CBV with Hb-200 significantly increased the CPV (73.7 +/- 1.8 mL/kg) when compared with the baseline level (53.5 +/- 1.8 mL/kg; n = 5; P < 0.05). The difference between the groups was first observed 15 minutes after infusion. CPV was significantly higher in study group 1 (87.0 +/- 2.2 mL/kg) compared with the other groups (80.2 +/- 2.6 mL/kg in the control group and 79.5 +/- 3.6 mL/kg in study group 2). After a second transfusion, the differences between the groups became more prominent. Study group 2 demonstrated the highest CPV and CBV. The two study groups demonstrated a statistically significant increase in circulating volumes compared with the control group. The magnitude of this difference was lower than expected based on in vitro colloid osmotic pressure measurements.

Purpose: Accuracy of measurement of low hemoglobin concentrations using the HemoCue, a B-hemoglobin photometer (HemoCue AB, Angelholm, Sweden) may exhibit significant variability. Infusion of hemoglobin-based oxygen carriers (HBOC) results in low concentrations of plasma hemoglobin. Our study assessed B-hemoglobin photometer measurement accuracy of three HBOC: (hemoglobin glutamer-200 (bovine; Oxyglobin, Biopure Corp., Cambridge, MA, USA); hemoglobin glutamer-250 (bovine; Hemopure, Biopure Corp, Cambridge, MA, USA), and hemoglobin-raffimer, (human; Hemolink, Hemosol, Inc., Toronto, Ontario, Canada)., Methods: In the laboratory, 45 split canine plasma samples were mixed with hemoglobin glutamer-200 (8.13, 16.25, 32.5 g x L(-1) concentrations), 45 samples were mixed with hemoglobin glutamer-250 (8.13, 16.25, 32.5 g x L(-1) concentrations), 45 with hemoglobin-raffimer (12.5, 25.0, 50.0 g x L(-1) concentrations), and measured. Plasma samples without HBOC served as control. Hemoglobin concentration was determined by a laboratory analyzer (Coulter Corporation, Hiafeah, FL, USA) and B-hemoglobin photometer (HemoCue, Angelholm, Sweden). Two independent technicians performed blinded sample measurements and randomly tested each sample five times. Results were analyzed according to Bland and Altman analysis., Results: B-hemoglobin photometer demonstrated high repeatability for all three HBOCs. Repeatability coefficients were 0.37 g x L(-1) and 0.48 g x L(-1) for hemoglobin glutamer-200, 0.39 g x L(-1) and 0.4 g x L(-1) for hemoglobin glutamer-250 and 1.07 g x L(-1) and 0.85 g x L(-1) for hemoglobin-raffimer. An acceptable agreement was found between the B-hemoglobin photometer and the laboratory analyzer for all three HBOCs tested., Conclusion: The B-hemoglobin photometer accurately determined the concentration of three HBOC solutions dissolved in canine plasma.

We present a simple modification of the iterative learning control algorithm of Arimoto et al. (1984) for the case where the inputs are bounded and time-rate-limited. The Jacobian error condition for monotonicity of input-error, rather than output-error, norms, is specified, the latter being insufficient to assure convergence, as proved herein. To the best of our knowledge, these facts have not been previously pointed out in the iterative learning control literature. We present a new proof that the modified controller produces monotonically decreasing input error norms, with a norm that covers the entire time interval of a learning trial.

Unlabelled: We sought to correlate in vivo microvascular, systemic function, hemodynamic, and oxygenation changes in autologous shed blood (n = 4) and hemoglobin glutamer-200 (Hb-200) (n = 4) resuscitations in hypovolemic dogs. Hemorrhage (approximately 40% blood loss) reduced mean arterial pressure to approximately 50 mm Hg and caused significant (P < 0.01) decreases in hematocrit, total hemoglobin, mean pulmonary arterial pressure, cardiac output, and oxygen delivery and significant (P < 0.01) increases in heart rate, systemic vascular resistance, and lactic acidosis. Significant (P < 0.01) changes in conjunctival microvascular variables also occurred, including a 19% decrease in venular diameter and 79% increase in average blood flow velocity. Shed blood resuscitation returned microvascular, systemic function, hemodynamic, and oxygenation variables to prehemorrhagic baseline values. In contrast, Hb-200 failed to restore hematocrit, total hemoglobin, cardiac output, oxygen delivery index, and systemic venous resistance to baseline, but it restored other systemic functions and all hemodynamic and microvascular changes. In addition, Hb-200 resuscitation in hypovolemic dogs (approximately 40% blood loss) did not cause extreme hemodilution or fatal outcome. This study confirms that real-time (in vivo) microvascular studies, which were conducted only in small rodent models in the past, can be performed simultaneously with systemic function, hemodynamic, and oxygenation studies in a large animal model for relevant data correlation., Implications: This is the first time that changes in the blood circulation have been studied, quantified, and correlated with systemic function, hemodynamic, and oxygenation changes in shock and during shock treatment in a large animal model. This study was performed by a new technology developed in-house to noninvasively and quantitatively study blood vessels in real time.

Rationale and Objectives: The authors tested whether noninvasive magnetic resonance (MR) oximetry is accurate in the in vivo measurement of oxygen saturation in a stroma-free, hemoglobin-based oxygen carrier (HBOC)., Materials and Methods: A central venous catheter was placed in the inferior vena cava (IVC) of 10 New Zealand white rabbits (weight range, 2.5-3.2 kg). Each rabbit underwent removal of 20% of blood volume followed by resuscitation with 10 mL/kg of bovine HBOC-200. Oxygen saturation of the blood mixture was measured in vivo at the IVC with MR oximetry, with separate in vitro calibration for each animal. Blood drawn from the IVC was measured with ex vivo oximetry, which was used as the standard of reference. The in vivo and ex vivo measurements were compared., Results: There was no significant difference (P > .1) between measurements obtained with MR oximetry and ex vivo oximetry. The results with in vivo MR oximetry demonstrated excellent correlation with those from ex vivo oximetry (r = 0.99) over a wide range of physiologic oxygen saturation values (16.7%-74.9%) in venous blood., Conclusion: Noninvasive in vivo MR measurement of oxygen saturation is valid for whole blood mixed with stroma-free hemoglobin. Therefore, MR oximetry may be clinically useful for assessing the oxygenation status in patients resuscitated with a HBOC.

The objective of this investigation was to study the effects of the first marketed haemoglobin-based oxygen carrier, Hemoglobin glutamer-200 (bovine) (Hb-200) (Oxyglobin) on splanchnic perfusion and oxygenation in a canine model of acute hypovolaemia. Twelve anaesthetized dogs [mean weight 30.8 (S.D. 1.4) kg] were instrumented for recordings of heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), cardiac output and cranial mesenteric arterial (CMA) and venous blood flows (CMV). Total and plasma haemoglobin (Hb), oxygen content and saturation, lactate concentration, pH and blood gases were analysed in arterial, mixed venous and mesenteric venous blood samples. Measurements were made before (baseline) and after 1 h of haemorrhage, after which animals were resuscitated with either shed blood (controls) or Hb-200 until HR, MAP and CVP returned to prehaemorrhage levels. Recordings were repeated immediately and 3 h after termination of fluid resuscitation, after which organ specimens were obtained for microscopic examination. Haemorrhage (average 32 ml kg(-1)) reduced MAP to 50 mm Hg, increased HR and systemic vascular resistance (SVR), and was accompanied in both the systemic and the splanchnic circulation by significant decreases in blood flow, Hb content and oxygen delivery (DO2), and lactic acidosis. In controls, all variables recovered to baseline after isovolaemic resuscitation with shed blood. In dogs resuscitated with a small volume of Hb-200 (10 ml kg(-1)), HR, MAP, CVP and CMA and CMV blood flows returned to baseline. However, cardiac output, total Hb, oxygen content and systemic and mesenteric DO2 remained depressed while SVR increased further. Mesenteric and systemic acid-base status recovered in both groups, and there was no difference in microscopic tissue damage between groups. Thus, Hb-200 reconstituted splanchnic perfusion and oxidative metabolism in spite of pronounced systemic vasoconstriction and insufficient restoration of CO and DO2; it may improve diffusive oxygen transport in the microvasculature by virtue of haemodilution and its high efficiency in the uptake and release of oxygen.

Unlabelled: Hemoglobin-based oxygen carriers (HBOC) may be ideal for monitoring circulating plasma volume (CV-P) and circulating blood volume (CV-B). We used an HBOC (Hemoglobin glutamer-200 [bovine], Oxyglobin; Biopure, Cambridge, MA) as an indicator for relative CV-B in the rabbit model. Accuracy of the technique was determined by comparison with the Evans blue dye (EBD) dilution technique in 19 anesthetized female New Zealand rabbits weighing 2.0 to 10.6 kg. The measurements were performed at baseline, after hemorrhage (1/3 of CV-B), normovolemic hemodilution (replacement of 1/3 CV-B by Hextend; Abbot Laboratories, North Chicago, IL), and hypervolemic hemodilution (additional infusion of Hextend(R) in a volume equal to 1/3 of CV-B). Hemoglobin concentration was measured by using a HemoCue photometer (HemoCue AB, Angelholm, Sweden). EBD concentration was analyzed by using linear regression to estimate Time 0 concentration; Time 0 was defined as EBD injection time. The difference between CV-P values determined by EBD and HBOC dilution was independent from the magnitude of the CV-P value. The relative bias was 1.29 mL, and the precision (one SD) was 2.82 mL. The difference did not reach statistical significance., Implications: Circulating plasma and blood volumes can be accurately estimated by plasma hemoglobin concentration measurements by using hemoglobin-based oxygen carrier infusion.

Rehabilitation robots are assistive devices designed for use by people with severe disability in order to gain independence in tasks of daily living. MANUS is a wheelchair-mounted general-purpose manipulator now in use with over 100 people in their homes in the Netherlands, in France and in other countries. MANUS has six main degrees of freedom, excluding the gripper. To keep the arm slim, the motors are placed in the main base. The resulting low mass of the arm ensures a reduced impulse in case of collision. A complex system of toothed belts, hollow shafts and geared wheels drives the end-effector. MANUS is equipped with a two-fingered gripper. The fingers have passive canting mechanisms which ensure a three-point grasping action for most objects. A modular control system is used for steering the manipulator to its desired position. For this, a simple but effective user interface has been developed which is configurable by the end-user as well as by the therapist.

This study was designed to validate in vitro oxygen saturation (SO2) measurements with the NOVA CO-Oximeter (Nova Biomedical Corp, Waltham, Mass, USA) in canine blood containing hemoglobin (Hb) glutamer-200 bovine (Hb-200; Oxyglobin, Biopure, Cambridge, Mass, USA) as a Hb-based oxygen carrier recently introduced into clinical practice. In the first set of experiments, stored blood from 6 mixed-breed canine blood donors was used. Target PO2 levels were reached in aliquots of blood samples by tonometry. Oxygen saturation was then measured with the test device and calculated based on known PO2 values. In the second set of experiments, total oxygen content was directly measured by means of an oxygen-specific electrode in aliquots of fresh whole arterial, venous, and mixed (arterial-venous) blood withdrawn from the same canine blood donors. Hb-200 was added to those blood samples to yield plasma Hb concentrations of 1.62, 3.25, 6.50, and 9.75 g/dL. Based on Hb content and SO2 measured by the NOVA CO-Oximeter in these samples, total oxygen content was also calculated for each sample and compared with measured values. A strong correlation was found between SO2 values measured with the co-oximeter in samples after tonometry, and calculated SO2 based on known PO2. Directly measured total blood O2 content varied by

Objective: The study was designed to validate oxygen saturation measurements from the NOVA CO-Oximeter, the i-STAT System, and the Corning 170 blood gas analyzer under conditions similar to the clinical application of a hemoglobin-based oxygen carrier., Design: A canine model was used for both in-vitro and in-vivo experiments., Setting: Canine laboratory for in-vivo, and tonometry laboratory for in-vitro sets., Participants: 6 mixed-breed canines, 30 kg; canine remainder sample blood., Interventions: In the first set of experiments, the target blood pO2 levels were reached by tonometry. In the second set of experiments, quantitative measurements of total oxygen content with the LEXO2Con-K, were performed immediately followed by measurements with the NOVA CO-Oximeter and the i-STAT system. HBOC was added in concentrations of 16.2, 32.5, 65.0, and 97.5 g/L. To analyze the clinical significance of the differences in the results obtained with the different investigated instruments; blood samples from dogs treated with HBOC after acute hemorrhagic shock were used., Results: There was a strong correlation between the oxygen saturation values measured with the investigated instruments in samples after tonometry and known oxygen partial pressure. The total calculated O2 content varied by 5% based on results generated by calculations based on the investigated instruments. The results did not change with different oxygenation of the sample. The differences among methods were not significant when HBOC concentration was 16.2 g/L. Higher concentrations of HBOC increased the difference between calculated and measured oxygen content; i-STAT system demonstrated a greater deviation from the results of the other two instruments. Systemic oxygen uptake based on investigated instruments showed high correlation with values based on LEXO2CON-K measurements (r=0.97 for CO-Oximeter, 0.96 for gas analyzer, and 0.79 for i-STAT). Systemic oxygen uptake values based on CO-Oximeter and blood gas analyzer data showed a 75% accuracy; i-STAT accuracy was 63% for control samples and 50% for samples after HBOC infusions., Conclusion: The NOVA CO-OXimeter is an accurate analyzer for measurement of oxygen saturation after HBOC infusion in the canine model. Use of the i-STAT system should be limited to arterial samples or for samples with a low HBOC concentration.

Activation of prejunctional beta-adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of beta-adrenoceptor involved. In [3H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the beta 2-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 microM). Isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin- and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained concentration elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the beta 1-selective antagonist 1-[2-((3-carbamoyl- 4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM). The results confirm the opposite beta-adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional beta-adrenoceptor is of the beta 2-subtype.

Effects of isoprenaline on contraction, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) as well as on contractions elicited by exogenous noradrenaline and ATP were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In [3H]-noradrenaline-pretreated tissues, electrical stimulation elicited an overflow of tritium and ATP and a biphasic contraction. Isoprenaline (1-100 nM) reduced the contraction, mainly phase I, and enhanced the evoked overflow of tritium: evoked overflow of ATP was not changed significantly. No, or almost no, contraction remained in [3H]-noradrenaline-pretreated tissues exposed to both prazosin (0.3 microM) and suramin (300 microM), and the evoked overflow of ATP was reduced by about 82%. Under these conditions, isoprenaline (1-100 nM) again enhanced the evoked overflow of tritium, but it now decreased the evoked overflow of ATP. Propranolol (1 microM), when added on top of prazosin and suramin, prevented the effects of isoprenaline (1-100 nM). In some tissues not pretreated with [3H]-noradrenaline, purinergic and adrenergic components of the neurogenic contraction (again to 210 pulses, 7 Hz) were isolated by exposure to prazosin (0.3 microM) and suramin (300 microM), respectively. Isoprenaline (1-100 nM) decreased the isolated purinergic component but did not change significantly the isolated adrenergic component. Contractions elicited by ATP (1000 microM) were not changed and contractions elicited by noradrenaline (100 microM) were slightly increased by isoprenaline (1-100 nM). Isoprenaline (100 nM) did not change the degradation of ATP (100 microM) by pieces of the vas deferens. It is concluded that, in the guinea-pig vas deferens, activation of prejunctional beta-adrenoceptors modulates the neural release of noradrenaline and ATP in opposite directions: release of noradrenaline is enhanced, whereas release of ATP is decreased.

Contractions, release of noradrenaline and release of ATP elicited by the indirectly acting sympathomimetic amine tyramine and responses elicited by exogenous noradrenaline were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In tissues pretreated with pargyline 1 mM, tyramine 300 microM, when added to the superfusion medium for 2 min, elicited contraction and an overflow of tritium (mainly [3H]-noradrenaline) and ATP. Contraction and ATP overflow responses were prevented and tritium overflow was greatly reduced by desipramine 10 microM. Prazosin 0.3 microM abolished contractions and evoked ATP overflow without changing tritium overflow. Blockade of postjunctional P2-purinoceptors by suramin 300 microM caused a marked decrease of tyramine-evoked contractions and a slight reduction of tritium overflow whereas evoked ATP overflow was markedly increased. The effect on contraction was not shared by two other P2-purinoceptor antagonists, namely pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) 32 microM and diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) 32 microM: PPADS increased contractions about fourfold, whilst DIDS had no effect at all. When the vas deferens was superfused for 24 min with medium containing tyramine 300 microM, evoked contractions and tritium overflow continued throughout whereas ATP overflow faded rapidly to basal values. In the presence of prazosin 0.3 microM, tyramine 300 microM again failed to elicit contractions as well as an overflow of ATP. Application of noradrenaline 10 microM instead of tyramine also resulted in prolonged contraction and an overflow of ATP that declined rapidly. It is concluded that all ATP released by tyramine is non-neuronal in origin, secondary to the activation of postjunctional alpha 1-adrenoceptors by released noradrenaline. The non-neural ATP does not seem to play a functional role in smooth muscle contraction and derives from a postjunctional source which is subject to a rapid depletion upon sustained alpha 1-adrenoceptor activation.

We studied some properties of the release of noradrenaline and ATP in isolated sympathetically innervated tissues. Release was elicited by electric stimulation and assessed as overflow of tritiated compounds (after labelling with [3H]noradrenaline) and enzymically measured ATP, respectively. Evans blue, which inhibits ectonucleotidases, greatly increased the evoked overflow of ATP, indicating that a major part of the ATP was metabolized after release. Much of the ATP was postjunctional in origin. The neural fraction was isolated when postjunctional release was suppressed by prazosin (alpha 1-adrenoceptor antagonist) and suramin (P2 purinoceptor antagonist). Comparison of neural ATP and [3H]-noradrenaline release showed that prostaglandin E2 reduced the release of both co-transmitters to a similar extent. Activation of prejunctional alpha 2-adrenoceptors, however, preferentially reduced the release of [3H]noradrenaline, and activation of prejunctional A1 purinoceptors reduced preferentially the release of ATP. Nucleotides such as ATP depressed the release of [3H]noradrenaline through two receptors: the well-known prejunctional A1 receptors and a separate group of prejunctional P2 purinoceptors. P2 antagonists increased the release of [3H]-noradrenaline. Overall, the results indicate differential storage, release and modulation of release of the two sympathetic co-transmitters. They also indicate that postganglionic sympathetic axons possess receptors for both co-transmitters: alpha 2 and P2 autoreceptors.

Contractions and overflow of tritium and ATP elicited by hypogastric nerve stimulation (HNS) and field stimulation (FS) were studied in the guinea-pig isolated vas deferens preincubated with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. HNS and FS elicited contraction, tritium overflow and ATP overflow. HNS at supramaximal current strength produced smaller responses than did FS at supramaximal current strength (210 pulses/7 Hz). Supramaximal HNS and submaximal FS were used in the remainder of the study. Prazosin (0.3 mumol/l) reduced contractions and the overflow of ATP elicited by both HNS and FS; the evoked overflow of tritium was not changed (210 pulses/7 Hz). Combined administration of prazosin (0.3 mumol/l) and suramin (300 mumol/l) abolished contractions and reduced the overflow of ATP elicited by both HNS and FS slightly more than did prazosin alone; tritium overflow again was not changed (210 pulses/7 Hz). Contractions, tritium overflow and ATP overflow increased with the frequency of both HNS and FS (from 7 to 25 Hz; 210 pulses); the increase in ATP overflow with frequency was more marked than the increase in tritium overflow. The preferential increase of ATP overflow with the frequency of HNS and FS persisted in the combined presence of prazosin (0.3 mumol/l) and suramin (300 mumol/l). The study confirms for HNS, a more physiologic way of sympathetic nerve stimulation, several observations previously obtained with FS. First, HNS-evoked ATP release is detectable as an overflow of ATP into the superfusion fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of Evans blue on nucleotide breakdown, nucleotide-evoked contractions and electrically evoked contractions, overflow of ATP and overflow of tritium (after labelling with [3H]-noradrenaline) was studied in rat vas deferens. Pieces of vas deferens degraded 83 to 85% of added ATP, ADP and 2-methyl-thio ATP (all 100 microM) over 30 min. Evans blue (100 microM) reduced this degradation to 22 to 26%. Nucleotides elicited contraction with potency declining in the order alpha,beta-methylene ATP > 2-methylthio ATP > ATP > ADP. Evans blue (100 microM) shifted the concentration-response curve of alpha, beta-methylene ATP to the right and increased the maximum. Concentration-response curves of ATP, ADP and 2-methylthio ATP, in contrast, were shifted to the left and responses were much potentiated. In the presence of Evans blue, the rank order of potency was ATP > 2-methylthio ATP > alpha, beta-methylene ATP > ADP. Electrical field stimulation (100 pulses at 10 Hz) elicited contraction and an overflow of tritium and ATP. Evans blue (100 microM) did not alter the contraction and the evoked overflow of tritium but increased 24-fold the evoked overflow of ATP. The results indicate that Evans blue may serve as an-albeit impure-ecto-nucleotidase inhibitor in functional experiments. Such experiments demonstrate that the low potency of ATP (and also ADP and 2-methylthio ATP) in eliciting contraction, and the small size of the overflow of ATP upon sympathetic nerve stimulation, are due to rapid breakdown.

Effects of angiotensin II and prostaglandin E2 on contractions, release of noradrenaline and release of ATP elicited by electrical stimulation (210 pulses, 7 Hz) were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In some experiments postsynaptic alpha 1-adrenoceptors and P2X-purinoceptors were blocked by prazosin and suramin, respectively, to isolate the neural fraction of the overflow of ATP. Electrical stimulation elicited an overflow of tritium and ATP and, in the absence of prazosin and suramin, contraction. In the absence of prazosin and suramin, angiotensin II (1-100 nM) enhanced contractions as well as the evoked overflow of tritium and ATP. All parameters were increased by about the same percentage for a given concentration of angiotensin II. The effect of prostaglandin E2 (1-100 nM) was complex. Contractions were mainly enhanced, the evoked overflow of tritium was reduced, whereas the evoked overflow of ATP was predominantly increased. No or almost no contraction remained in the presence of prazosin and suramin, and the evoked overflow of ATP was decreased to about 16%. Angiotensin II (1-100 nM) again enhanced the evoked overflow of tritium and ATP. Both were increased by about the same percentage for a given concentration of angiotensin II and also were increased by about the same percentage as obtained in the absence of prazosin and suramin.(ABSTRACT TRUNCATED AT 250 WORDS)

The purinergic response of the guinea-pig vas deferens to long trains of pulses at high frequency consists of an initial twitch followed by a much lower plateau. Mechanical, neurochemical and electrophysiological techniques were used to examine the reason for the fade. Mechanical measurements. In tissues stimulated by trains of 180 pulses/10 Hz and treated with prazosin to suppress the noradrenergic contraction component, the response to alpha, beta-methylene ATP and to exogenous ATP was as high during the secondary plateau of the purinergic neurogenic contraction as it was outside electrical stimulation periods; the response to 50 pulses/100 Hz was also unchanged during the low plateau. The plateau was not increased by reactive blue 2,8-(p-sulphophenyl)theophylline, propranolol or capsaicin. Neurochemical measurements. In tissues preincubated with [3H]-noradrenaline, electrical stimulation elicited an overflow of tritium and of ATP. In the absence of drugs as well as in the presence of prazosin and suramin to suppress contractions, the overflow of tritium per pulse decreased slightly in the course of trains of 90 pulses/10 Hz; the overflow of ATP per pulse decreased to a greater extent on average, but the decrease was not statistically significant. In the presence of prazosin and nifedipine, also to suppress contractions, the overflow of tritium per pulse again decreased slightly in the course of trains of 105 pulses/10 Hz, but the overflow of ATP per pulse if anything tended to increase. Electrophysiological measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release. In tissues preincubated with [3H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 microM and even more so by prazosin 0.3 microM combined with suramin 300 microM. They were also reduced by nifedipine 10 microM and even more so by nifedipine 10 microM combined with ryanodine 20 microM (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [3H]-noradrenaline, exogenous noradrenaline 10 microM and alpha,beta-methylene ATP 10 microM elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 microM but not suramin 300 microM and were greatly reduced by nifedipine 10 microM and in Ca(2+)-free medium. Responses to alpha,beta-methylene ATP were blocked by suramin 300 microM but not prazosin 0.3 microM, were reduced by nifedipine 10 microM (effect on ATP overflow not significant) and were reduced even more in Ca(2+)-free medium. Neuropeptide Y 0.3 microM caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of P1-purinoceptor activation on contractions, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) was studied in the superfused vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. In the absence of other drugs, adenosine (10-100 microM) did not change evoked contractions but reduced the evoked overflow of tritium and ATP. In subsequent experiments alpha 1-adrenoceptors were blocked by prazosin, P2-purinoceptors by suramin and alpha 2-adrenoceptors by rauwolscine. No or almost no contraction remained under these conditions. The evoked overflow of tritium was 505% and the evoked overflow of ATP 34% of that observed in the absence of prazosin, suramin and rauwolscine. Adenosine (1-100 microM) again reduced the evoked overflow of tritium and ATP, and so did the A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.032-0.32 microM). Adenosine and CCPA decreased the evoked overflow of ATP to a greater extent than the evoked overflow of tritium. It is concluded that neural release of both postganglionic sympathetic cotransmitters, noradrenaline and ATP, is decreased upon activation of prejunctional P1- (A1-) purinoceptors in guinea-pig vas deferens. The A1-receptor-mediated inhibition of the release of ATP is more marked than the inhibition of the release of noradrenaline, a pattern opposite to the inhibition produced by activation of prejunctional alpha 2-autoreceptors.

The release of both sympathetic co-transmitters, noradrenaline and ATP, is modulated via presynaptic receptors. However, the degree of the modulation may differ indicating that the ratio of the released co-transmitters changes upon presynaptic receptor activation. For example, alpha 2-autoinhibition affects the release of noradrenaline more markedly than the release of ATP. Some sympathetic axon terminals possess presynaptic P2-purinoceptors which are activated by endogenous ATP. These receptors are a novel kind of auto-receptor: they mediate a presynaptic negative feedback mechanism in which released ATP inhibits subsequent co-transmitter release.

Contractions, release of previously stored [3H]-noradrenaline (measured as overflow of total tritiated compounds) and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) were studied in the superfused vas deferens of the guinea pig. Prazosin and suramin were used to suppress non-neural ATP release, and effects of bromoxidine and rauwolscine on the neural release thus isolated were examined. Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. Both prazosin (0.03-3 microM) and suramin (30-300 microM) reduced contractions as well as the evoked overflow of ATP. No visible contraction remained in 21 of 28 tissues exposed to prazosin 0.3 microM combined with suramin 300 microM. The evoked overflow of ATP under these conditions was about 17% of that observed in the absence of drugs. In the presence of prazosin 0.3 microM and suramin 300 microM, bromoxidine (0.01-1 microM) decreased and rauwolscine (0.1-10 microM) increased the evoked overflow of both tritium and ATP. Rauwolscine increased the evoked overflow of tritium to a significantly greater extent than the overflow of ATP. It is concluded that the overflow of ATP elicited by electrical (neural) stimulation in the presence of prazosin 0.3 microM and suramin 300 microM reflects purely neural release of ATP. This release of ATP, like the release of noradrenaline, is modulated through prejunctional alpha 2-adrenoceptors. The alpha 2-adrenoceptor modulation of the release of noradrenaline seems to be more marked than the modulation of the release of ATP.

1. Tramadol is a centrally acting analgesic with low opioid receptor affinity and, therefore, presumably additional mechanisms of analgesic action. Tramadol and its main metabolite O-desmethyltramadol were tested on rat central noradrenergic neurones of the nucleus locus coeruleus (LC), which are involved in the modulation of nociceptive afferent stimuli. 2. In pontine slices of the rat brain the spontaneous discharge of action potentials of LC cells was recorded extracellularly. (-)-Tramadol (0.1-100 microM), (+)-tramadol (0.1-100 microM), (-)-O-desmethyl-tramadol (0.1-100 microM) and (+)-O-desmethyltramadol (0.01-1 microM) inhibited the firing rate in a concentration-dependent manner. (+)-O-desmethyltramadol had the highest potency, while all other agonists were active at a similar range of concentrations. 3. (-)-Tramadol (10, 100 microM) was less inhibitory in brain slices of rats pretreated with reserpine (5 mg kg-1, 5 h before decapitation) than in controls. 4. The effect of (-)-tramadol (10 microM) was abolished in the presence of the alpha 2-adrenoceptor antagonist, rauwolscine (1 microM), whilst that of (+)-O-desmethyltramadol (0.3 microM) virtually disappeared in the presence of the opioid antagonist, naloxone (0.1 microM). (+)-Tramadol (30 microM) and (-)-O-desmethyl-tramadol (10 microM) became inactive only in the combined presence of naloxone (0.1 microM) and rauwolscine (1 microM). 5. In another series of experiments, the membrane potential of LC neurones was determined with intracellular microelectrodes. (-)-Tramadol (100 microM) inhibited the spontaneous firing and hyper-polarized the cells; this effect was abolished by rauwolscine (1 microM). (+)-O-desmethyltramadol (10 microM)had a similar but somewhat larger effect on the membrane potential than (-)-tramadol. The (+)-O-desmethyltramadol-(10 microM) induced hyperpolarization was abolished by naloxone (0.1 microM).6. The hyperpolarizing effect of noradrenaline (30 microM) was potentiated in the presence of (-)-tramadol(100 microM), but not in the presence of (+)-O-desmethyltramadol (10 microM). There was no potentiation of the noradrenaline (30 microM) effect, when the cells were hyperpolarized by current injection to an extent similar to that produced by (-)-tramadol (100 microM).7. Both noradrenaline (100 microM) and (- )-tramadol (100 microM) decreased the input resistance.8. The results confirm that the analgesic action of tramadol involves both opioid and non-opioid components. It appears that (-)-tramadol inhibits the uptake of noradrenaline and via a subsequent increase in the concentration of endogenous noradrenaline indirectly stimulates alpha2-adrenoceptors. (+)-0-desmethyltramadol seems to stimulate directly opioid micro-receptors. The effects of (+)-tramadol and(-)-O-desmethyltramadol consist of combined micro-opioid and alpha2-adrenergic components.

1. Effects of opioid agonists on the purinergic and adrenergic components of neurogenic contractions and in some experiments on transmitter overflow were studied in the mouse isolated vas deferens. 2. When the vas deferens was stimulated every 2 min by pairs of pulses 2 s apart in the presence of prazosin 0.3 microM (to isolate the purinergic component) or alpha,beta-methylene-ATP 3 microM (to isolate the adrenergic component), each pulse elicited a separate twitch. The opioid agonists [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO, mu-receptor-selective), [D-Pen2,D-Pen5]enkephalin (DPDPE, delta-selective) and trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide (U-50488, kappa-selective) concentration-dependently reduced both purinergic and adrenergic contractions. For each agonist, maximal effects and concentrations causing half-maximal effects were very similar for inhibition of the purinergic component on the one hand and for inhibition of the adrenergic component on the other hand, although the adrenergic component was inhibited with a slight preference. Moreover, effects on contractions elicited by the first and the second pulse of the pairs were very similar. 3. When vasa deferentia preincubated with [3H]-noradrenaline were stimulated with trains of 100 pulses delivered at 20 Hz, morphine 10 microM reduced significantly both evoked tritium overflow and evoked contractions. Its effect was antagonized by naloxone. 4. It is concluded that, in contrast to drugs acting at some other presynaptic receptors, opioid mu-, delta- and kappa-agonists inhibit purinergic and adrenergic neurogenic contractions of the mouse vas deferens in a similar manner. In contrast to a previous report, no enhancement by morphine of the release of noradrenaline elicited by high frequency pulse trains was observed.