TNF-alpha augments pulmonary vasoconstriction via the inhibition of nitrovasodilator activity. We tested the hypothesis that tumor necrosis factor-alpha (TNF-alpha) increases pulmonary vasoconstriction by decreases in nitric oxide- (NO) dependent vasodilation. Lungs were isolated from guinea pigs 18 h after intraperitoneal injection of either TNF-alpha (1.60 x 10(5) U/kg) or control. U-46619 (365 mM/min) caused increases in pulmonary arterial and capillary pressures, pulmonary arterial and venous resistances, and lung weight. TNF-alpha augmented the U-46619-induced increases in pulmonary arterial and capillary pressures, pulmonary arterial and venous resistances, and lung weight. Methylene blue (1 microM), which inhibits the activation of soluble guanylate cyclase by NO, had an effect similar to TNF-alpha on the pulmonary response to U-46619 alone but was not additive to the effect of TNF-alpha. NG-monomethyl-L-arginine (270 microM), an inhibitor of NO generation, also enhanced the response to U-46619. Lung effluent levels of nitrite, the oxidation product of NO, were reduced after treatment with either TNF-alpha or NG-monomethyl-L-arginine compared with U-46619 alone. In addition, lungs isolated after TNF-alpha treatment showed decreased vasodilation in response to acetylcholine (10(-8)-10(-5) M) compared with control; however, vasodilation in response to L-arginine (10 mM) and nitroprusside (10(-6.3) and 10(-6) M), agents that promote NO release, was not decreased in TNF-alpha-treated lungs. The data indicate that TNF-alpha induces an increase in vascular constriction in response to U-46619 and a decrease in vasodilation in response to acetylcholine. The mechanism for the TNF-alpha-induced alteration in pulmonary vascular reactivity may be decreased generation of NO.


Increased thrombin-antithrombin III complexes after 1 h of physical exercise. Concentrations of thrombin-antithrombin III (TAT) complexes in plasma were previously reported to be increased after a 100-km run, while fibrinopeptide A (FPA) concentration remained unchanged. Thus, antithrombin III appears to neutralize thrombin generated during running and prevents fibrin formation. To determine the clinical relevance of these findings, we compared the effects of exhaustive running (1 h, n = 10) on the plasma concentrations of prothrombin fragments F1 and F2, TAT, FPA, and beta-thromboglobulin with the effects of recreational jogging (1 h, n = 10) and exhaustive bicycling on an ergometer (1 h, n = 8). Prothrombin fragments F1 and F2 and TAT concentrations increased significantly in each group. The most significant increase in TAT concentration was measured in the running group (from 1.72 +/- 0.49 to 3.61 +/- 1.03 ng/ml, P < 0.001). The best correlation was found between the postexercise TAT and lactate concentrations (r = 0.62, n = 28, P < 0.001). Mean FPA concentrations after exercise did not exceed normal values in any of the three groups analyzed. An increase in beta-thromboglobulin concentration was measured in the running and in the cycling group. Thus, thrombin is formed, in particular, when associated with anaerobic metabolism, and platelets are activated during high-intensity exercise.


Intrapleural fluid movements described by a porous flow model. We injected technetium-labeled albumin (at a concentration similar to that of the pleural fluid) in the costal region of anesthetized dogs (n = 13) either breathing spontaneously or apneic. The decay rate of labeled activity at the injection site was studied with a gamma camera placed either in the anteroposterior (AP) or laterolateral (LL) projection. In breathing animals (respiratory frequency approximately 10 cycles/min), 10 min after the injection the activity decreased by approximately 50% on AP and approximately 20% on LL imaging; in apneic animals the corresponding decrease in activity was reduced to approximately 15 and approximately 3%, respectively. We considered label translocation from AP and LL imaging as a result of bulk flows of liquid along the costomediastinal and gravity-dependent direction, respectively. We related intrapleural flows to the hydraulic pressure gradients existing along these two directions and to the geometry of the pleural space. The pleural space was considered as a porous medium partially occupied by the mesh of microvilli protruding from mesothelial cells. Solution of the Kozeny-Carman equation for the observed flow velocities and pressure gradients yielded a mean hydraulic radius of the pathways followed by the liquid ranging from 2 to 4 microns. The hydraulic resistivity of the pleural space was estimated at approximately 8.5 x 10(5) dyn.s.cm-4, five orders of magnitude lower than that of interstitial tissue.


Training can improve muscle strength and endurance in 78- to 84-yr-old men. Nine men, 78-84 yr of age, participated in a dynamometer training program 2-3 times/wk, totaling 25 sessions, using voluntary maximal isometric, concentric, and eccentric right knee-extension actions (30 and 180 degrees/s). Measurements of muscle strength with a Kin-Com dynamometer and simultaneous electromyograms (EMG) were performed of both sides before and after the training period. Muscle biopsies were taken from the right vastus lateralis muscle. The total quadriceps cross-sectional area was measured with computerized tomography. Training led to an increase in maximal torque for concentric (10% at 30 degrees/s) and eccentric (13-19%) actions in the trained leg. The EMG activity increased at maximal eccentric activities. The total cross-sectional quadriceps area of the trained leg increased by 3%, but no changes were recorded in muscle fiber areas in these subjects, who already had large mean fiber areas (5.15 microns 2 x 10(3)). The fatigue index measured from 50 consecutive concentric contractions at 180 degrees/s decreased and the citrate synthase activity increased in all but one subject. The results demonstrate that increased neural activation accompanies an increase in muscle strength at least during eccentric action in already rather active elderly men and that muscle endurance may also be improved with training.


Effects of hypoxia on the discharge of group III and IV muscle afferents in cats. The reflex pressor response evoked by static muscular contraction is widely believed to be caused by the stimulation of group III and IV afferents. Although the specific nature of the contraction-induced stimulus to these thin-fiber afferents is unknown, they are thought to be stimulated in part by a condition arising from a mismatch between blood supply and demand in the exercising muscle. Hypoxia, a condition found in skeletal muscle during such a mismatch, may stimulate these afferents. We have therefore tested the hypothesis that perfusion of the triceps surae muscles with hypoxic blood stimulates group III and IV afferents in barbiturate-anesthetized cats. We found that 3-3.5 min of hypoxia with the triceps surae muscles at rest significantly (P < 0.05) increased the average discharge rate of contraction-sensitive group IV afferents but had no effect on the average discharge rate of contraction-sensitive group III afferents. Hypoxia had only trivial effects on the discharge of contraction-insensitive group III and IV afferents. Hypoxia stimulated 4 of 11 contraction-sensitive group IV afferents and 2 of 13 contraction-sensitive group III afferents. The responses of the afferents stimulated by hypoxia were small in magnitude. Hypoxia with the muscles at rest appeared to have no effect on either hydrogen or lactate ion concentrations in the femoral venous blood. In addition, hypoxia increased the responses to contraction in only 3 of 22 group III and 4 of 21 group IV afferents tested. We conclude that muscle tissue hypoxia is a minor stimulus to afferents that sense a mismatch between blood supply and demand during static contraction.


Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle. Mouse extensor digitorum longus (EDL) muscle was subjected to a dose of gamma irradiation that causes reproductive death of satellite cells and/or to chronic compensatory overload, achieved by removal of the distal portion of the tibialis anterior muscle. Four weeks later the mass, fiber type percentage, and fiber size of the EDL muscle were measured. Both the irradiated + overloaded and the irradiated only EDL muscles were significantly lighter and contained significantly smaller fibers than untreated muscle or muscle subjected to chronic overload only. Overload muscle, whether irradiated or not, had a larger percentage of type IIx fibers and a smaller percentage of type IIb fibers than muscle that had not been overloaded. The results confirm that satellite cell proliferation is a prerequisite for muscle hypertrophy induced by synergist incapacitation, but it appears not to be required for the maintenance of, or change in, normal muscle fiber myosin heavy chain phenotype expression.


Vasoconstrictor-mediated release of lactate from the perfused rat hindlimb. The effects of different vasomodulators on lactate release by the constant-flow-perfused rat hindlimb were examined and compared with that by perfused mesenteric artery, incubated preparations of aortas, soleus and epitrochlearis muscles, and perifused soleus muscles. Infusion of vasopressin (0.5 nM), angiotensin II (5 nM), norepinephrine (50 nM), and methoxamine (10 microM) into the hindlimbs of 180- to 200-g rats increased the perfusion pressure by 112-167% from 30.4 +/- 0.8 mmHg, O2 consumption by 26-68% from 6.4 +/- 0.2 mumol.g-1 x h-1, and lactate efflux by 148-380% from 5.41 +/- 0.25 mumol.g-1 x h-1. Hindlimbs of 100- to 120-g rats responded similarly to angiotensin II. Isoproterenol (1 microM) had no effect on O2 uptake or perfusion pressure but increased lactate release by 118%. Nitroprusside (0.5 mM) markedly inhibited the vasoconstrictor-mediated increases in lactate release, perfusion pressure, and O2 consumption by the hindlimb but had no effect on isoproterenol-mediated lactate efflux. Serotonin (6.7 microM) increased lactate release from the perfused mesenteric artery by 120% from 5.48 mol.g-1 x h-1. Lactate release by incubated aorta was increased by angiotensin II (50 nM), isoproterenol (1 microM), and mechanical stretch. The increase mediated by angiotensin II was blocked by glycerol trinitrate (2.2 microM), which had no effect on lactate release by isoproterenol. Neither angiotensin II (5 nM) nor vasopressin (0.5 nM) increased lactate release from incubated soleus and epitrochlearis muscles; however, lactate release was increased by isoproterenol, and this increase was unaffected by glycerol trinitrate (2.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)


Vascular effects of platelet-activating factor in lambs: role of cyclo- and lipoxygenase. In adult sheep, platelet-activating factor (PAF) effects include systemic hypotension and pulmonary hypertension. To identify developmental differences in vascular responses to PAF, we studied the effects of C18- and C16-PAF in 49 +/- 2- (SE) day-old lambs. Responses of upstream (arteries and microvessels) and venous segments of the lung to C18-PAF were determined both in vivo and in isolated lungs. In isolated lungs, the role of eicosanoids in PAF effects was also determined. In vivo, both C18- and C16-PAF caused a significant increase in systemic and pulmonary vascular resistance. The magnitude of vascular responses to C16-PAF was greater than that to C18-PAF. C18-PAF constricted both upstream and venous segments of the pulmonary circulation. Cyclooxygenase inhibition in isolated lungs attenuated arterial constriction to C18-PAF, whereas simultaneous cyclooxygenase and lipoxygenase inhibition completely blocked the effects of C18-PAF. In summary, in contrast to PAF effects in adult sheep, PAF constricts both systemic and pulmonary vessels in lambs, with significant pulmonary venous constriction. Eicosanoids, especially lipoxygenase products, play a major role in mediating PAF effects in the lung.


Evaluation of human dynamic contraction by phonomyography. Phonomyogram (PMG, or acoustic myogram) is known to increase with force in isometric contractions. We investigated this relationship for dynamic contractions against different inertias. PMG and surface electromyogram (EMG) from biceps brachii and brachioradialis muscles were simultaneously recorded with the angular acceleration of elbow flexions. These were self-initiated movements (30 degrees) toward a fixed target and performed against two different inertias. PMG and EMG were integrated from the onset of the signal to the end of the acceleration phase. Phono- and electromechanical delays were also measured. For integrated EMG (iEMG), there was a linear relationship between integrated PMG (iPMG) and force, the slope of which did not depend on inertia. There was also a linear relationship between iPMG or iEMG and angular acceleration, with a higher slope for the highest inertia condition. There was also a family of linear relationships between iPMG or iEMG and angular acceleration, and their slopes depended on inertia. Measurements of the phono- and electromechanical delays showed that onset of PMG followed that of EMG but preceded onset of acceleration. It is suggested that PMG expresses tension of the underlying muscle contractile elements. Given the simplicity of the PMG method, we conclude that PMG allows convenient evaluation of muscle tension during human dynamic contraction.


Operation Everest II: metabolic and hormonal responses to incremental exercise to exhaustion. The reasons for the reduced exercise capacities observed at high altitudes are not completely known. Substrate availability or accumulations of lactate and ammonium could have significant roles. As part of Operation Everest II, peak oxygen uptakes were determined in five normal male volunteers with use of progressively increasing cycling work loads at ambient barometric pressures of 760, 380, and 282 Torr. Decrements from sea level (SL) to 380 and 282 Torr occurred in peak power output (19 and 47%), time to exhaustion (19 and 48%), and oxygen uptake (41 and 61%), respectively. Arterial saturations after exhaustive exercise were decreased to 63% at 380 Torr and 39% at 282 Torr. At 380 and 282 Torr, postexercise plasma concentrations of glucose and free fatty acids were not increased, whereas plasma glycerol concentrations were decreased relative to SL (145 +/- 24 microM at 380 Torr and 77 +/- 10 microM at 282 Torr vs. 213 +/- 24 microM at SL). Preexercise plasma insulin concentrations were elevated at both 380 and 282 Torr (87 +/- 16 pM at 380 Torr and 85 +/- 18 pM at 282 Torr vs. 41 +/- 30 pM at SL). In general, postexercise concentrations of plasma catecholamines were decreased at altitude compared with SL. Preexercise lactate and ammonium concentrations were not different at any simulated altitude. From these data neither substrate availability nor metabolic product accumulation limited exercise capacity at extreme simulated altitude.


Lactate exchange and removal abilities in sickle cell patients and in untrained and trained healthy humans. Arterial blood lactate concentrations obtained on seven black males with hemoglobin sickle cell disease (SC) before, during, and after graded bicycle exercise up to exhaustion were compared with those of seven untrained (HU) and seven trained (HT) healthy males of the same ethnic origin. Lactate recovery curves were fitted by a biexponential time function consisting of a rapidly increasing and a slowly decreasing component. Higher work rates were reached by the HU and HT than by the SC group. Blood lactate rose distinctly over the corresponding preexercise resting values after the 25-, 50-, and 100-W exercise steps for the SC, HU, and HT groups, respectively. The arterial oxygen content was significantly lower for the SC than for the HU group at rest and at the end of exercise. The velocity constants of the slowly decreasing component of the lactate recovery curves were similar for the SC, HU, and HT groups despite the fact that they cycled up to different absolute work rates. The velocity constant of the rapidly increasing component was significantly higher for the HT. In terms of the functional meaning given to these constants and in view of their inverse relationship with absolute work rate (Freund et al. J. Appl. Physiol. 61: 932-939, 1986), these results indicate that, relative to the HU, the HT and the SC display improved and impaired abilities, respectively, to exchange and to remove lactate.


Relationship between arterial oxygen desaturation and ventilation during maximal exercise. The purpose of the present study was to investigate the contribution of ventilation to arterial O2 desaturation during maximal exercise. Nine untrained subjects and 22 trained long-distance runners [age 18-36 yr, maximal O2 uptake (VO2max) 48-74 ml.min-1 x kg-1] volunteered to participate in the study. The subjects performed an incremental exhaustive cycle ergometry test at 70 rpm of pedaling frequency, during which arterial O2 saturation (SaO2) and ventilatory data were collected every minute. SaO2 was estimated with a pulse oximeter. A significant positive correlation was found between SaO2 and end-tidal PO2 (PETO2; r = 0.72, r2 = 0.52, P < 0.001) during maximal exercise. These statistical results suggest that approximately 50% of the variability of SaO2 can be accounted for by differences in PETO2, which reflects alveolar PO2. Furthermore, PETO2 was highly correlated with the ventilatory equivalent for O2 (VE/VO2; r = 0.91, P < 0.001), which indicates that PETO2 could be the result of ventilation stimulated by maximal exercise. Finally, SaO2 was positively related to VE/VO2 during maximal exercise (r = 0.74, r2 = 0.55, P < 0.001). Therefore, one-half of the arterial O2 desaturation occurring during maximal exercise may be explained by less hyperventilation, specifically for our subjects, who demonstrated a wide range of trained states. Furthermore, we found an indirect positive correlation between SaO2 and ventilatory response to CO2 at rest (r = 0.45, P < 0.05), which was mediated by ventilation during maximal exercise. These data also suggest that ventilation is an important factor for arterial O2 desaturation during maximal exercise.


Nitrogen tensions in brachial vein blood of Korean ama divers. Intravascular bubble formation and symptoms of decompression sickness have been reported during repetitive deep breath-hold diving. Therefore we examined the pattern of blood N2 kinetics during and after repetitive breath-hold diving. To study muscle N2 uptake and release, we measured brachial venous N2 partial pressure (PN2) in nine professional Korean breath-hold divers (ama) during a 3-h diving shift at approximately 4 m seawater depth and up to 4 h after diving. PN2 was determined with the manometric Van Slyke method. Diving time and depth were recorded using a backpack computer-assisted dive longer that allowed calculating the surface-to-depth time ratio to derive the effective depth. With the assumption that forearm muscle N2 kinetics follow the general Haldanian principles of compression and decompression, i.e., forearm muscle is a single compartment with a uniform tissue PN2 equal to venous PN2, PN2 data were fitted to monoexponential functions of time. In the early phase of the diving shift, PN2 rapidly increased to 640 Torr (half time = 6 min) and then slowly declined to baseline levels (half time = 36 min) after the work shift. Peak PN2 levels approximated the alveolar PN2 derived from the effective depth. We conclude that forearm muscle N2 kinetics are well described by a Haldanian single-compartment model. Decompression sickness is theoretically possible in the ama; it did not occur because the absolute PN2 remained low due to the shallow working depth of the ama we studied.


Adaptation to fatigue of long duration in human wrist movements. Subjects made fast, accurate, consistent wrist flexions under normal conditions and under conditions of low-frequency fatigue. Movements made 1 h after fatiguing exercise were indistinguishable from those made before exercise, even though twitch tensions were only approximately 60% of their fresh values. Electromyograms (EMGs) recorded from the fatigued muscles were, however, different from those recorded before exercise. EMGs during unfatigued movements showed multiple bursts typical for rapid movements. In the presence of low-frequency fatigue, the duration of the first burst was longer than that under normal conditions, and its onset occurred earlier relative to the initiation of movement. The area of the second agonist burst and, in some cases, the antagonist burst, was increased, although changes in their timings were unclear. We conclude that subjects adapted to low-frequency fatigue by changing the neural patterns controlling their muscles and present a simple model of excitation-contraction coupling that demonstrates how the observed changes in excitation can produce the same kinematics.


Role of platelet-activating factor in the ovine heparin-protamine reaction. Platelet-activating factor (PAF) infusion into sheep, as well as protamine reversal of heparin anticoagulation, causes thromboxane release into plasma, pulmonary hypertension, hypoxemia, and leukopenia. We investigated the possible role of PAF in the heparin-protamine reaction. Intravenous protamine was administered to neutralize heparin anticoagulation in five awake sheep and caused an increase of mean pulmonary arterial pressure from 16.6 +/- 1 (SE) mmHg at base-line to 47 +/- 9 mmHg at 1 min after protamine injection (P < 0.01) because of a 4.5-fold increase of pulmonary vascular resistance. This neutralization reaction induced a 25% reduction of circulating leukocyte count and arterial PO2. Undetectable blood levels of PAF were measured by bioassay and high-performance liquid chromatography during these heparin-protamine reactions. Infusion of BN 52021 (20 mg/kg), a PAF receptor antagonist, before rechallenging the same sheep with heparin and then protamine did not reduce the level of peak pulmonary hypertension or the degree of hypoxemia and leukopenia. We conclude that the leukopenia and thromboxane-mediated pulmonary vasoconstriction occurring after rapid intravascular formation of heparin-protamine complexes in sheep are not due to the release of PAF.


Development of pulmonary intravascular macrophage function in newborn lambs. We sought to determine whether pulmonary intravascular macrophages are involved in pulmonary vascular sensitivity to intravenously injected particles in sheep. We estimated that newborn lambs have few of these macrophages at birth but develop a 10-fold greater density within 2 wk. Awake, chronically instrumented newborn lambs showed no change in pulmonary vascular driving pressure (pulmonary arterial minus left atrial pressure) after injection of either liposomes [2 +/- 3 (SD) cmH2O; n = 5] or Monastral blue particles (3 +/- 2 cmH2O; n = 6) and showed no net pulmonary production of thromboxane B2, the stable metabolite of the vasoconstrictor thromboxane A2. In contrast, five of those lambs 2 wk later showed both an increase in pulmonary vascular driving pressure after injection of liposomes and Monastral blue (20 +/- 16 and 25 +/- 15 cmH2O, respectively; P < 0.05) and net pulmonary production of thromboxane B2 (171 +/- 103 and 429 +/- 419 pg/ml plasma, respectively; P < 0.05). Older lambs (n = 5) had higher pulmonary uptakes than newborn lambs (n = 6) of radioactive liposomes (47 +/- 13 vs. 12 +/- 10%; P < 0.01) and Monastral blue (53 +/- 6 vs. 21 +/- 10%; P < 0.05). We conclude that pulmonary intravascular macrophages are responsible for the sensitivity of sheep to intravenous foreign particles and are essential for a cascade of processes leading to microvascular injury.


Separate effects of ischemia and reperfusion on vascular permeability in ventilated ferret lungs. In systemic organs, ischemia-reperfusion injury is thought to occur during reperfusion, when oxygen is reintroduced to hypoxic ischemic tissue. In contrast, the ventilated lung may be more susceptible to injury during ischemia, before reperfusion, because oxygen tension will be high during ischemia and decrease with reperfusion. To evaluate this possibility, we compared the effects of hyperoxic ischemia alone and hyperoxic ischemia with normoxic reperfusion on vascular permeability in isolated ferret lungs. Permeability was estimated by measurement of filtration coefficient (Kf) and osmotic reflection coefficient for albumin (sigma alb), using methods that did not require reperfusion to make these measurements. Kf and sigma alb in control lungs (n = 5), which were ventilated with 14% O2-5% CO2 after minimal (15 +/- 1 min) ischemia, averaged 0.033 +/- 0.004 g.min-1.mmHg-1.100 g-1 and 0.69 +/- 0.07, respectively. These values did not differ from those reported in normal in vivo lungs of other species. The effects of short (54 +/- 9 min, n = 10) and long (180 min, n = 7) ischemia were evaluated in lungs ventilated with 95% O2-5% CO2. Kf and sigma alb did not change after short ischemia (Kf = 0.051 +/- 0.006 g.min-1.mmHg-1.100 g-1, sigma alb = 0.69 +/- 0.07) but increased significantly after long ischemia (Kf = 0.233 +/- 0.049 g.min-1 x mmHg-1 x 100 g-1, sigma alb = 0.36 +/- 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of ventilation inhomogeneity on DLcoSB-3EQ in normal subjects. In patients with airflow obstruction, we found that ventilation inhomogeneity during vital capacity single-breath maneuvers was associated with decreases in the three-equation single-breath CO diffusing capacity of the lung (DLcoSB-3EQ) when breath-hold time (tBH) decreased. We postulated that this was due to a significant resistance to diffusive gas mixing within the gas phase of the lung. In this study, we hypothesized that this phenomenon might also occur in normal subjects if the breathing cycle were altered from traditional vital capacity maneuvers to those that increase ventilation inhomogeneity. In 10 normal subjects, we examined the tBH dependence of both DLcoSB-3EQ and the distribution of ventilation, measured by the mixing efficiency and the normalized phase III slope for helium. Preinspiratory lung volume (V0) was increased by keeping the maximum end-inspiratory lung volume (Vmax) constant or by increasing V0 and Vmax. When V0 increased while Vmax was kept constant, we found that the tBH-independent and the tBH-dependent components of ventilation inhomogeneity increased, but DLcoSB-3EQ was independent of V0 and tBH. Increasing V0 and Vmax did not change ventilation inhomogeneity at a tBH of 0 s, but the tBH-dependent component decreased. DLcoSB-3EQ, although independent of tBH, increased slightly with increases in Vmax. We conclude that in normal subjects increases in ventilation inhomogeneity with increases in V0 do not result in DLcoSB-3EQ becoming tBH dependent.


Topography of cat medullary ventral surface hypoxic acidification. The topographic relationship between previously identified medullary ventral surface respiratory chemosensitive regions and brain surface extracellular fluid (ECF) acid production during acute hypoxia was explored in anesthetized, paralyzed, and artificially ventilated cats. Glass pH electrodes (0.8-mm diam, sheathed in stainless steel tubing) were mounted in mechanical contact with surfaces of medullary surface or adjacent pyramids, pons, spinal cord, or parietal cortex. Isocapnic hypoxia of 5 min [at arterial O2 saturation (SaO2) = 48 +/- 10%] reduced pH over rostral (Mitchell) and caudal (Loeschcke) areas by 0.12 +/- 0.09 and 0.07 +/- 0.04, respectively (n = 10, P < 0.05). Change in pH (delta pH) was proportional to desaturation with slopes 100 delta pH/delta SaO2 of 0.45 (rostral) and 0.20 (caudal) (R = 0.91 and 0.88, respectively). pH drop usually began within 3 min of hypoxia, became stable between 5 and 15 min, began to rise within 2 min of reoxygenation, and returned to control within 10 min. During equally hypoxic tests, intermediate area (Schläfke), pons, and spinal cord surfaces showed no significant acid shift. Parietal cortex ECF pH dropped more slowly but steadily by 0.079 +/- 0.034 during 20 min at SaO2 = 50% after a small but significant initial alkaline shift, and acidification of cortical surface continued for > 5 min after reoxygenation. We conclude that medullary ventral chemosensitive regions produce more lactic acid during hypoxia than neighboring brain surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)


Internal carotid arterial flow velocity during exercise in Tibetan and Han residents of Lhasa (3,658 m). Cerebral blood flow increases with acute exposure to high altitude, but the effect of hypoxia on the cerebral circulation at rest and during exercise appears influenced by the duration of high-altitude exposure. To determine whether internal carotid artery flow velocity increased with exercise in long-term residents of high altitude and whether resting values and the response to exercise differed in lifelong vs. acclimatized newcomer male residents of high altitude, we studied 15 native Tibetan and 11 Han ("Chinese") 6 +/- 2-yr residents of Lhasa (3,658 m), Tibet Autonomous Region, China. Noninvasive Doppler ultrasound was used to measure internal carotid artery diameter, mean flow velocity, and, in combination, hemoglobin and arterial O2 saturation to assess cerebral O2 delivery. Tibetan and Han groups were similar in body size and resting internal carotid artery diameter, blood pressure, hemoglobin concentration, internal carotid artery mean flow velocity, and calculated cerebral O2 delivery. Submaximal exercise increased internal carotid artery mean flow velocity and cerebral O2 delivery in the Tibetan and Han subjects. At peak exercise, the Tibetans sustained the increase in flow velocity and cerebral O2 delivery, whereas the Hans did not. Across all exercise levels up to and including peak effort, the Tibetans demonstrated a greater increase in internal carotid artery flow velocity and cerebral O2 delivery relative to resting values than did the Hans. The greater cerebral O2 delivery was accompanied by increased peak exercise capacity in the Tibetan compared with the Han group. Our findings suggest that the cerebral blood flow response to exercise is maintained in Tibetan lifelong residents of high altitude.


Time course of respiratory mechanics during histamine challenge in the dog. We studied the dynamics of respiratory mechanical parameters in anesthetized tracheostomized paralyzed dogs challenged with a bolus of histamine injected either venously (venous group) or arterially (arterial group). The venous group was further divided into two groups: the first was bilaterally vagotomized and received hexamethonium bromide (denervated group), and the second also received atropine sulfate (atropine group). In the venous group, tissue resistance (Rti) and tissue elastance (Eti) increased biphasically, whereas airway resistance was monophasic and synchronized with the second rise of the tissue parameters. In the arterial group, Rti, Eti, and airway resistance increased synchronously. The denervated and atropine groups showed dynamics similar to those of the venous group. We postulate that the first phase observed in Rti and Eti in the venous group is due to constriction of the smooth muscles of the peripheral airways and blood vessels distorting the parenchyma. The second and larger phase is then due to histamine reaching the bronchial circulation and constricting the central airways, again distorting the parenchyma. The results from the arterial group support this hypothesis, whereas those from the denervated group ascertain that none of the phases observed in the venous group was due to nervous reflexes.


Canine pulmonary filtration coefficient calculated from optical, radioisotope, and weight measurements. Three independent methods were used to estimate filtration coefficient (Kf) in isolated dog lungs perfused with low-hematocrit (Hct) blood. Pulmonary vascular pressure was increased by 12-23 cmH2O to induce fluid filtration. Average Kf (ml.min-1 x cmH2O-1 x 100 g dry wt-1) for six lungs was 0.26 +/- 0.05 (SE) with use of equations describing conservation of optically measured protein labeled with indocyanine green. Good agreement was found when a simplified version of the multiequation theory was applied to the data (0.24 +/- 0.05). Both optical estimates were lower than those predicted by constant slope (0.55 +/- 0.07) or extrapolation (1.20 +/- 0.15) techniques, which are based on changes in total lung weight. Subsequent studies in five dog lungs investigated whether the higher Kf from weight analyses could be caused by prolonged pulmonary vascular filling. We found that 51Cr-labeled red blood cells (RBCs), monitored over the lung, continued to accumulate for 30 min after vascular pressure elevations of 9-16 cmH2O.Kf was determined by subtracting computed vascular filling from total weight change (0.28 +/- 0.06) and by perfusate Hct changes determined from radiolabeled RBCs (0.23 +/- 0.04). These values were similar to those obtained from analysis of optical data with the complete model (0.30 +/- 0.06), the simplified version (0.26 +/- 0.05), and from optically determined perfusate Hct (0.16 +/- 0.03). However, constant slope (0.47 +/- 0.04) and extrapolation (0.57 +/- 0.07) computations of Kf were higher than estimates from the other methods. Our studies indicate that prolonged blood volume changes may accompany vascular pressure elevations and produce overestimates of Kf with standard weight measurement techniques. However, Kf computed from optical measurements is independent of pulmonary blood volume changes.


Reduced vascular responsiveness after a single bout of dynamic exercise in the conscious rabbit. We measured agonist-induced changes in the iliac artery blood flow velocity (IFV) independent of baroreflex-mediated compensatory mechanisms in chronically instrumented New Zealand White rabbits (n = 8). Animals were instrumented with a Doppler flow probe around the right common iliac artery. A Teflon catheter was inserted into the right iliolumbar artery for local infusion of the vasoactive agonists. Another Teflon catheter was inserted in the left femoral artery for the measurement of pulsatile and mean arterial (MAP) blood pressures and heart rate (HR). The alpha-adrenergic receptor agonist phenylephrine (PE, 1.32-10.0 micrograms), the beta 1- and beta 2-adrenergic receptor agonist isoproterenol (IP, 0.022-0.11 micrograms), and the purinergic receptor agonist adenosine (AD, 10.0-100.0 micrograms) were injected into the functionally isolated hindlimb, and dose-response curves were generated. Changes in IFV were obtained without changes in MAP or HR. Exercise increased HR, MAP, and IFV (65.3 +/- 7.1 beats/min, 11.1 +/- 2.2 mmHg, and 2.2 +/- 0.3 kHz, respectively). The maximum responses to PE, AD, and IP were reduced 29.0 +/- 6.7, 50.7 +/- 8.5, and 61.0 +/- 8.1%, respectively, after exercise. In conclusion, exercise attenuated adrenergic and purinergic receptor-mediated vascular responses in the intact conscious rabbit.


Effects of varied air velocity on sweating and evaporative rates during exercise. This study was designed to determine the extent to which changes in the evaporative power of the environment (Emax) affect sweating and evaporative rates. Six male subjects undertook four 60-min bouts of cycle ergometer exercise at 56% maximal O2 uptake (VO2max).Emax was varied by differences in ambient temperature and airflow; two exercise bouts took place at 24 degrees C and two at 35 degrees C, with air velocity at < 0.2 and 3.0 m/s in both. Total sweat production was estimated from body weight loss, whereas whole body evaporative rate was measured continuously from a Potter beam balance. Body core temperature was measured continuously from a thermocouple in the esophagus (T(es)), with mean skin temperature (Tsk) computed each minute from thermocouples at eight sites. Total body sweat loss was significantly greater (P < 0.05) in the 0.2- than in the 3.0-m/s condition at both 24 and 35 degrees C. Tsk was higher (P < 0.05) in the still-air conditions at both temperatures, but final T(es) was significantly higher (P < 0.05) in still air only in the 35 degrees C environment. Thus the reduced Emax in still air caused a greater heat storage, thereby stimulating a greater total sweat loss. However, in part because of reduced skin wettedness, the slope of the sweat rate-to-T(es) relation at 35 degrees C in the 3.0-m/s condition was 118% that at 0.2 m/s (P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)


Dynamic response of the isolated passive rat diaphragm strip. To further our understanding of the mechanisms underlying chest wall mechanics, we investigated the dynamic response of the isolated passive rat diaphragm strip. Stress adaptation of the tissue was measured from 0.05 to 60 s after subjecting the strips to strain steps of normalized strain amplitudes from 0.005 to 0.04. The tissue resistance (R), elastance (E), and hysteresivity (eta) were measured in the same range of amplitudes by sinusoidally straining the strip at frequencies from 0.03125 to 10 Hz. The stress (T) depended exponentially on the strain (epsilon) and relaxed and recovered linearly with the logarithm of time. E increased linearly with the logarithm of frequency and decreased with increasing amplitude. R fell hyperbolically with frequency and showed an amplitude dependence similar to that of E. To interpret the strong nonlinear behavior, we extended the viscoelastic model of Hildebrandt (J. Appl. Physiol. 28: 365-372, 1970) to include an exponential stress-strain relationship. Accordingly, the step response was described by T - Tr = Tr(e alpha delta epsilon - 1)(1 - gamma log t), where delta epsilon is the strain amplitude, Tr is the initial operating stress, alpha is a measure of the stress-strain nonlinearity, and gamma is the rate of stress adaptation. The oscillatory response of the model was computed by applying Fung's quasi-linear viscoelastic theory. This quasi-linear viscoelastic model fitted the step and oscillatory data fairly well but only if alpha depended negatively on delta epsilon, as might be expected in a plastic material.


Cardiovascular responses to lower body negative pressure in trained and untrained older men. To determine whether aerobic conditioning alters the orthostatic responses of older subjects, cardiovascular performance was monitored during graded lower body negative pressure in nine highly trained male senior athletes (A) aged 59-73 yr [maximum O2 uptake (VO2 max) = 52.4 +/- 1.7 ml.kg-1 x min-1] and nine age-matched control subjects (C) (VO2 max = 31.0 +/- 2.9 ml.kg-1 x min-1). Cardiac volumes were determined from gated blood pool scintigrams by use of 99mTc-labeled erythrocytes. During lower body negative pressure (0 to -50 mmHg), left ventricular end-diastolic and end-systolic volume indexes and stroke volume index decreased in both groups while heart rate increased. The decreases in cardiac volumes and mean arterial pressure and the increase in heart rate between 0 and -50 mmHg were significantly less in A than in C. For example, end-diastolic volume index decreased by 32 +/- 4 ml in C vs. 14 +/- 2 ml in A (P < 0.01), mean arterial pressure declined 7 +/- 5 mmHg in C and increased by 5 +/- 3 mmHg in A (P < 0.05), and heart rate increased 13 +/- 3 beats/min in C and 7 +/- 1 beats/min in A (P < 0.05). These data suggest that increased VO2 max among older men is associated with improved orthostatic responses.


Altitude acclimatization and energy metabolic adaptations in skeletal muscle during exercise. To determine whether the working muscle is able to sustain ATP homeostasis during a hypoxic insult and the mechanisms associated with energy metabolic adaptations during the acclimatization process, seven male subjects [23 +/- 2 (SE) yr, 72.2 +/- 1.6 kg] were given a prolonged exercise challenge (45 min) at sea level (SL), within 4 h after ascent to an altitude of 4,300 m (acute hypoxia, AH), and after 3 wk of sustained residence at 4,300 m (chronic hypoxia, CH). The prolonged cycle test conducted at the same absolute intensity and representing 51 +/- 1% of SL maximal aerobic power (VO2 max) and between 64 +/- 2 (AH) and 66 +/- 1% (CH) at altitude was performed without a reduction in ATP concentration in the working vastus lateralis regardless of condition. Compared with rest, exercise performed during AH resulted in a greater increase (P < 0.05) in muscle lactate concentration (5.11 +/- 0.68 to 22.3 +/- 6.1 mmol/kg dry wt) than exercise performed either at SL (5.88 +/- 0.85 to 11.5 +/- 3.1) or CH (5.99 +/- 0.88 to 12.4 +/- 2.1). These differences in lactate concentration have been shown to reflect differences in arterial lactate concentration and glycolysis (Brooks et al. J. Appl. Physiol. 71: 333-341, 1991). The reduction in glycolysis at least between AH and CH appears to be accompanied by a tighter metabolic control. During CH, free ADP was lower and the ATP-to-free ADP ratio was increased (P < 0.05) compared with AH.(ABSTRACT TRUNCATED AT 250 WORDS)


Energetics of walking and running: insights from simulated reduced-gravity experiments. On Earth, a person uses about one-half as much energy to walk a mile as to run a mile. On another planet with lower gravity, would walking still be more economical than running? When people carry weights while they walk or run, energetic cost increases in proportion to the added load. It would seem to follow that if gravity were reduced, energetic cost would decrease in proportion to body weight in both gaits. However, we find that under simulated reduced gravity, the rate of energy consumption decreases in proportion to body weight during running but not during walking. When gravity is reduced by 75%, the rate of energy consumption is reduced by 72% during running but only by 33% during walking. Because reducing gravity decreases the energetic cost much more for running than for walking, walking is not the cheapest way to travel a mile at low levels of gravity. These results suggest that the link between the mechanics of locomotion and energetic cost is fundamentally different for walking and for running.


Adaptation of rat skeletal muscle to creatine depletion: AMP deaminase and AMP deamination. AMP deaminase catalyzes deamination of the AMP formed in contracting muscles to inosine 5'-monophosphate (IMP). Slow-twitch muscle has only approximately 30% as high a level of AMP deaminase activity as fast-twitch muscle in the rat, and rates of IMP formation during intense contractile activity are much lower in slow-twitch muscle. We found that feeding the creatine analogue beta-guanidinopropionic acid (beta-GPA) to rats, which results in creatine depletion, causes a large decrease in muscle AMP deaminase. This adaptation was used to evaluate the role of AMP deaminase activity level in accounting for differences in IMP production in slow-twitch and fast-twitch muscles. beta-GPA feeding for 3 wk lowered AMP deaminase activity in fast-twitch epitrochlearis muscle to a level similar to that found in the normal slow-twitch soleus muscle but had no effect on the magnitude of the increase in IMP in response to intense contractile activity. Despite a similar decrease in ATP in the normal soleus and the epitrochlearis from beta-GPA-fed rats, the increase in IMP was only approximately 30% as great in the soleus in response to intense contractile activity. These results demonstrate that the accumulation of less IMP in slow- compared with fast-twitch skeletal muscle during contractile activity is not due to the lower level of AMP deaminase in slow-twitch muscle.


Retinoblastoma gene product Rb accumulates during myogenic differentiation and is deinduced by the expression of SV40 large T antigen. The cell growth suppression activity of the retinoblastoma gene (RB) product Rb is considered to be regulated by phosphorylation, whereas no positive correlation has been demonstrated between the activity and the amount of Rb protein in numbers of cell types examined. Both the RB mRNA and the protein were dramatically induced during terminal differentiation of the mouse skeletal muscle cell line C2 and its transfectant C2SVTts11, which stably harbors the SV40 T antigen gene linked to an inducible promoter. They were gradually deinduced when the terminally differentiated C2SVTs11 myotubes reentered the cell cycle through the induction of the large T antigen. Thus, the accumulation of Rb protein is likely to be required for growth arrest during differentiation of at least these myogenic cells, which have low basal levels of the protein.


Amino acid sequence of the 20-kDa regulatory light chain of porcine aorta media smooth muscle myosin. The amino acid sequence of the 20-kDa regulatory light chain (LC20) of myosin from porcine aorta media smooth muscle was determined. The LC20 consisted of 171 amino acid residues and its N-terminal Ser residue was blocked by an acetyl group. The amino acid sequence was identical with that of chicken gizzard myosin LC20 except that the 60th residue, Met in chicken gizzard LC20, was substituted for Leu in porcine aorta LC20.


Proteinase-sensitive sites on isolated rabbit dystrophin. Dystrophin was isolated from the purified large oligomeric dystrophin complex with its associated proteins (DC) of rabbit skeletal muscle by alkaline dissociation followed by gel filtration to remove the associated proteins. Isolated dystrophin and DC were subjected to digestion with calpain or alpha-chymotrypsin, and the generated polypeptide fragments were studied by immunoblot analysis using seven kinds of antibodies raised against antigens corresponding to various regions from the N- to the C-terminal of human dystrophin. For some fragments, the amino acid sequences at the N-termini were determined. Two proteinases, which bear distinct specificities, generated very similar fragments from purified dystrophin with or without the associated proteins. The cleavage sites found by mapping the fragments onto the dystrophin molecule were similar to those found in a previous study using crude mouse muscle cell membrane fraction [Koenig, M. & Kunkel, L.M. (1990) J. Biol. Chem. 265, 4560-4566]. On the basis of these results, we concluded that dystrophin has several unique proteinase-sensitive sites.


Possible involvement of clathrin in neuritogenesis induced by a protease inhibitor (benzyloxycarbonyl-Leu-Leu-Leu-aldehyde) in PC12 cells. Our previous reports showed that benzyloxycarbonyl (Z)-Leu-Leu-Leu-al (ZLLLal) induces neurite outgrowth in PC12 cells, and that 33-, 35-, and 180-kDa proteins from PC12 cells elute specifically from a Leu-Leu-Leu-al (LLLal)-coupled affinity column. Several lines of evidence suggest that the 33-, 35-, and 180-kDa proteins are components of clathrin, well-known for its role in endocytosis. Separation of clathrin into its heavy and light chains showed that the clathrin heavy chains have the ability to bind to a LLLal affinity column directly. Furthermore, ZLLLal enhances the rate of polymerization of clathrin triskelion to the coat structure. ZLLL-COOH does not cause neurite outgrowth in PC12 cells, and has no effect on the rate of clathrin polymerization. On immunocytochemical analysis of PC12 cells with an anti-clathrin heavy chain antibody, enhanced staining of the clathrin heavy chain was observed concomitantly with neurite outgrowth initiated by ZLLLal, but not by NGF. This study provides new insights into both the role of the clathrin molecule and the regulatory mechanism of neurite outgrowth.


Purification and characterization of novel sulfotransferase obtained from Klebsiella K-36, an intestinal bacterium of rat. A novel type of sulfotransferase was purified from Klebsiella K-36, an intestinal bacterium of rat. The enzyme (M(r) 160,000) is composed of two subunits (M(r) 73,000) with pI and optimal pH values of 5.3 and 10-10.5, respectively. The apparent Km for PNS (p-nitrophenyl sulfate) using phenol as an acceptor and that for phenol using PNS as a donor substrate were determined to be 0.11 and 0.66 mM, respectively. The enzyme is activated by magnesium ion and inhibited by EDTA.


Characterization of two novel pyruvylated glycosphingolipids containing 2'-aminoethylphosphoryl(-->6)-galactose from the nervous system of Aplysia kurodai. Two novel acidic glycosphingolipids containing pyruvylated galactose were purified from the nervous tissue of Aplysia kurodai by successive Iatrobeads column chromatographies. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structures of these acidic glycosphingolipids, named F-9 and FGL-I, were determined to be: [3,4-O-(S-1-carboxyethylidene)]Gal beta 1-->3 GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2] (2-aminoethylphosphoryl 1-->6)Gal beta 1-->4Glc beta 1-->1ceramide and [3,4-O-(S-1-carboxyethylidene)] Gal beta 1-->3GalNAc alpha 1-->3(Fuc alpha 1-->2)(2-aminoethylphosphonyl-->6 Gal beta 1-->4Glc beta 1-->1ceramide, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, pyruvylated glycosphingolipids containing phosphoethanolamine in addition to or in place of 2-aminoethylphosphonate are present in the nervous system of Aplysia.


Decreased arachidonate metabolism in mouse peritoneal macrophages after foam cell transformation with oxidized low-density lipoproteins. Oxidized low density lipoproteins (LDL) are now considered to be one of the atherogenic lipoproteins in vivo and to play an important role in the pathogenesis of atherosclerosis. We previously demonstrated in mouse peritoneal macrophages that oxidized LDL stimulated prostaglandin (PG) E2 synthesis when incorporated into the cells [Yokode, M. et al. (1988) J. Clin. Invest. 81, 720-729]. In this study, we investigated arachidonate metabolism in macrophages after foam cell transformation. The cells were incubated with 100 micrograms/ml of oxidized LDL for 18 h, then stimulated with zymosan. Lipid-enriched macrophages which had taken up oxidized LDL produced much less eicosanoids, such as PGE2, 6-keto-PGF1 alpha, and leukotriene C4 than control cells. After labeling of the cells with [14C]arachidonic acid, they were stimulated with zymosan and the phospholipase activity was determined. The activity of lipid-enriched cells was about two-thirds of that of control cells. Then we investigated the fatty acid composition of their phospholipid fraction to clarify arachidonic acid content and mobilization. Percent of arachidonic acid of lipid-enriched cells decreased and less arachidonic acid mobilization was observed after stimulation with zymosan. These data suggest that impaired arachidonate metabolism in lipid-enriched macrophages can be explained by their decreased phospholipase activity and changes in their fatty acid composition.


Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47, and its purification. Sphingomyelinase (sphingomyelin cholinephosphohydrolase) [EC 3.1.4.12] of Bacillus cereus was overproduced in a protein-hyperproducing strain, B. brevis 47, by cloning the gene into an expression vector pNU211, which has been developed to express a foreign gene utilizing a promoter and a signal sequence of an outer cell wall protein gene. From 1 liter of culture, about 10 mg of protein was purified to near-homogeneity by two steps of column chromatography; this is almost 500 times higher production compared to the conventional preparation from the original strain, B. cereus IAM 1208. The N-terminal amino acid sequence of the secreted enzyme was identical to that of the authentic enzyme, indicating that the signal sequence for secretion of B. cereus was processed properly in B. brevis 47.


Effect of the potential triplex DNA region on the in vitro expression of bacterial beta-lactamase gene in superhelical recombinant plasmids. The effect of a pyrimidine/purine-biased stretch which has the potential to form an unusual triplex DNA structure on gene expression has been analyzed by measuring the activity of beta-lactamase as a reporter gene in recombinant plasmids. The Escherichia coli transformant carrying the plasmid p7ERS which has a potential triplex DNA region expressed about twofold more beta-lactamase activity than that carrying the plasmid pUC19. Since the expression of beta-lactamase has been shown to be affected by template topology in vitro, this in vivo observation suggests that the inserted pyrimidine/purine-biased stretch modulates the topology of flanking regions by forming unusual DNA structure to keep the template at the superhelicity favorable for the expression of beta-lactamase.


Human T cell L-plastin bundles actin filaments in a calcium-dependent manner. The amino acid sequences deduced from cDNA analyses revealed that human leucocyte L-plastin phosphorylated in response to interleukin 1, 2 closely resembles a chicken intestinal microvilli protein, fimbrin, that bundles actin filaments [de Arruda et al. (1990) J. Cell Biol. 111, 1069-1079]. In the present work, it was observed that unphosphorylated L-plastin isolated from human T cells bundled F-actin just as fimbrin does. L-Plastin acted on T cell beta-actin, but hardly acted on muscle alpha-actin or chicken gizzard gamma-actin, whereas fimbrin bundled muscle alpha-actin. Unlike fimbrin, L-plastin's actin-bundling action was strictly calcium-dependent: the bundles were formed at pCa 7, but not at pCa 6. Under suitable conditions, approximately one molecule of L-plastin bound to 8 molecules of actin monomer in the actin filament.


A novel dihydrodiol dehydrogenase in bovine liver cytosol: purification and characterization of multiple forms of dihydrodiol dehydrogenase. Three enzymes (DD1, DD2, and DD3) having dihydrodiol dehydrogenase activity were purified to homogeneity from bovine cytosol. DD1 and DD2 were identified as 3 alpha-hydroxysteroid dehydrogenase and high-Km aldehyde reductase, respectively, as judged from their molecular weights, substrate specificities and inhibitor sensitivities. DD3 was a unique enzyme which could specifically catalyze the dehydrogenation of trans-benzenedihydrodiol and trans-naphthalenedihydrodiol without any activity toward the other tested alcohols, aldehydes, ketones, and quinones. The Km value of DD3 (0.18 mM) for benzenedihydrodiol was lower than those of other dihydrodiol dehydrogenases so far reported. DD3 immunologically crossreacted with DD1, but showed no crossreactivity with DD2. Additionally, DD3 was inhibited in a competitive manner, with a low Ki value of 1 microM, by androsterone, which was a good substrate for DD1. It was assumed that DD3 is a novel enzyme which is specific to dihydrodiols, exhibiting similarity to DD1 in immunological and structural properties.


Molecular cloning of cDNAs for rat proteasomes: deduced primary structures of four other subunits. Proteasomes (multi-protease complexes) are composed of approximately 15 non-identical subunits of similar sizes (molecular weight = 21-32 kDa), but different charges (isoelectric point = 4-9). Previously, we deduced the primary structures of 6 subunits of rat proteasomes by recombinant DNA techniques. In this paper we report the nucleotide sequences of 4 other subunits, rIOTA, rZETA, rDELTA, and rRING12, determined from cDNA clones isolated by screening a rat H4TG hepatoma cell cDNA library with the cDNAs of their human counterparts as probes. The polypeptides deduced from their nucleotide sequences consisted of 246, 241, 202, and 219 amino acid residues with calculated molecular weights of 27,399, 26,391, 21,649, and 23,324, and calculated isoelectric points of 6.37, 4.65, 4.84, and 4.70, respectively. These results and previous findings indicate that the primary structures of the subunits of rat proteasomes show considerably high inter-subunit homology, but can be classified into apparently distinct sub-groups, suggesting that rat proteasome genes form a multi-gene family with the same evolutionary origin, but have diverged during evolution to acquire possibly subunit-specific functions.


In vitro formation of estrogen receptor-heat shock protein 90 complexes. We previously showed that the 9 S estrogen receptor can be reconstituted from purified vero ER (estradiol binding subunit) and purified hsp 90 (heat shock protein 90) in vitro [Inano, K. et al. (1990) FEBS Lett. 267, 157-159]. In this study, we further characterized our reconstitution system to investigate the mechanism underlying the formation of 9 S ER. When a vero ER preparation stored at 4 degrees C for more than 20 h after affinity chromatography was used for the reconstitution of 9 S ER, 0.5 M NaSCN was essential, but not Na2MoO4 or other reagents. When, however, vero ER was used within 3 h after dissociation from an affinity resin, 9 S ER could be reconstituted in a relatively high yield without NaSCN. Moreover, if such a fresh vero ER preparation was used, 9 S ER could be reconstituted in the absence of NaSCN from not only unoccupied vero ER but also the occupied form. From these results it was suggested that the conformation of purified vero ER tends to change quickly in a time dependent manner, and so a chemical perturbant, NaSCN, is generally necessary for the reconstitution of 9 S ER from purified vero ER and purified hsp 90. The concentration of hsp 90 required for the reconstitution was only about 1.0 microM, which was lower than its physiological concentration. Based on these results, the mechanism underlying the formation of 9 S ER was discussed.


Three-dimensional structure of soybean beta-amylase determined at 3.0 A resolution: preliminary chain tracing of the complex with alpha-cyclodextrin. The three-dimensional structure of a complex of soybean beta-amylase [EC 3.2.1.2] with an inhibitor, alpha-cyclodextrin, has been determined at 3.0 A resolution by X-ray diffraction analysis. Preliminary chain tracing showed that the enzyme folded into large and small domains. The large domain has a (beta alpha)8 super-secondary structure, while the smaller one is formed from two long loops extending from the beta 3 and beta 4 strands of the (beta alpha)8 structure. The interface of the two domains together with shorter loops from the (beta alpha)8 structure form a deep cleft, in which alpha-cyclodextrin binds slightly away from the center. Two maltose molecules also bind in the cleft. One shares a binding site with alpha-cyclodextrin and the other is situated more deeply in the cleft.


Calphobindins (placental annexins) inhibit protein kinase C. Calphobindins (CPBs, placental annexins) are intracellular Ca(2+)- and phospholipid-dependent proteins like protein kinase C [EC 2.7.1.37]. We investigated the inhibitory effects of calphobindins on the protein kinase C activity in vitro. CPB I inhibited the protein kinase C activity for both histone phosphorylation and lipocortin phosphorylation, but CPB II and CPB III inhibited only the protein kinase C activity for histone phosphorylation. In the case of histone phosphorylation, all CPBs inhibited the protein kinase C activity in a concentration-dependent manner, and the IC50 (concentration required for 50% inhibition) value of CPB I was 70 nM. The inhibition of protein kinase C by CPB I was Ca(2+)-dependent, and did not disappear upon increasing the concentration of phosphatidyl-serine. Kinetic analysis by double-reciprocal plots indicated that CPB I interacted not only with phosphatidylserine but also with protein kinase C. Although CPB I partially interacts with phospholipid, it is conceivable that the inhibitory action of CPB I on protein kinase C results from direct interaction of CPB I with protein kinase C. Since CPBs are mainly present under the plasma membrane, it is presumed that CPB I is an endogenous inhibitor of protein kinase C, and according to intracellular circumstances, CPB II and CPB III may also be endogenous inhibitors.


Identification of the disulfide bonds in human plasma protein SP-40,40 (apolipoprotein-J). SP-40,40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40,40 were determined. SP-40,40 was purified from human seminal plasma by affinity chromatography using an anti-SP-40,40 monoclonal antibody and reversed-phase, high-performance liquid chromatography (HPLC). The protein was digested with trypsin and the fragments were separated by reversed-phase HPLC. The peptides containing disulfide bonds were fluorophotometrically detected with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The peptides containing more than two disulfide bonds were further digested with Staphylococcus aureus V8 protease and lysylendopeptidase, and the fragments were isolated by HPLC. The amino acid compositions and the amino acid sequences of the peptides containing only a disulfide bond were determined. Disulfide bonds thus determined were between Cys58(alpha)-Cys107(beta), Cys68(alpha)-Cys99(beta), Cys75(alpha)-Cys94(beta), and Cys86(alpha)-Cys80(beta). Since there was no free sulfhydryl groups in the SP-40,40 molecule, Cys78(alpha) and Cys91(beta) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in SP-40,40 are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of SP-40,40.


Fatty acid composition and properties of the liver microsomal membrane of rats fed diets enriched with cholesterol. Male rats were fed diets containing olive (OO) or evening primrose (EPO) oil (10% w/w), with or without added cholesterol (1% w/w). After 6-week feeding, the lipid and fatty acid compositions, fluidity, and fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both the OO and EPO diets, without added cholesterol, increased the contents of oleic and arachidonic acids, respectively, of rat liver microsomes. The results were consistent with the increases in delta 9 and delta 6 desaturation of n-6 essential fatty acids and the lower microviscosity in the EPO group. Dietary cholesterol led to an increase in the cholesterol content of liver microsomes as well as that of phosphatidylcholine (PC). The cholesterol/phospholipid and PC/PE (phosphatidylethanolamine) ratios were also elevated. Fatty acid composition changes were expressed as the accumulation of monounsaturated fatty acids, with accompanying milder depletion of saturated fatty acids in rat liver microsomes. In addition, the arachidonic acid content was lowered, with a concomitant increase in linoleic acid, which led to a significant decrease in the 20:4/18:2 ratio in comparison to in animals fed the cholesterol-free diets. Cholesterol feeding also increased delta 9 desaturase activity as well as membrane microviscosity, whereas it decreased delta 6 and delta 5 desaturase activities. There was a very strong correlation between fluidity and the unsaturation index reduction in the membrane. Furthermore, the activity of hydroxymethylglutaryl-CoA reductase increased and the activity of acyl-CoA:cholesterol acyltransferase decreased in liver microsomes from both cholesterol-fed groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Muscle actin cleaved by proteinase K: its polymerization and in vitro motility. Skeletal muscle actin was lightly digested by proteinase K, which cleaved the peptide bond between Met-47 and Gly-48, producing a C-terminal 35 kDa fragment. Proteinase K-cleaved actin (proK-actin) did not polymerize into F-actin upon addition of salt. In the presence of phalloidin, however, it polymerized slowly into F-actin (proK-F-actin), indicating that the cleaved actin did not dissociate into the individual cleaved fragments but retained the global structure of actin. Electron microscopy showed that proK-F-actin had the typical double-stranded structure of a normal actin filament and formed the arrowhead structure when decorated with HMM. Heavy meromyosin ATPase was weakly activated by proK-F-actin: Vmax = 0.24 s-1, and Kapp = 2.8 microM, while Vmax = 7.6 s-1, and Kapp = 13 microM by F-actin. Correspondingly, in vitro this proK-F-actin slid very slowly on HMM attached to a glass surface at an average velocity of 0.47 microns/s, or 1/12 of that of intact F-actin. The fraction of sliding filaments was less than 50%. Assuming that the nonmotile filaments attached to HMM were not involved in ATPase activation, the sliding velocity correlated with the ATPase activity activated by proK-F-actin.


A comparison of the fatigue behavior of human trabecular and cortical bone tissue. The fatigue properties of trabecular bone tissue (single trabeculae) and similarly sized cortical bone specimens from human tibia were experimentally determined on a microstructural level using four-point bending cyclic tests, and they were compared based on modulus, mineral density, and microstructural characteristics. The results showed that trabecular specimens had significantly lower moduli and lower fatigue strength than cortical specimens, despite their higher mineral density values. Fracture surface and microdamage analyses illustrated different fracture and damage patterns between trabecular and cortical bone tissue, depending upon their microstructural characteristics. Based on the results from mechanical tests and qualitative observations, a possible mechanical role of the cement lines in trabecular tissue microfracture was suggested.


Biomechanical analyses of rising from a chair. Quantification of the biomechanical factors that underlie the inability to rise from a chair can help explain why this disability occurs and can aid in the design of chairs and of therapeutic intervention programs. Experimental data collected earlier from 17 young adult and two groups of elderly subjects, 23 healthy and 11 impaired, rising from a standard chair under controlled conditions were analyzed using a planar biomechanical model. The joint torque strength requirements and the location of the floor reaction force at liftoff from the seat in the different groups and under several conditions were calculated. Analyses were also made of how body configurations and the use of hand force affect these joint torques and reaction locations. In all three groups, the required torques at liftoff were modest compared to literature data on voluntary strengths. Among the three groups rising with the use of hands, at the time of liftoff from the seat, the impaired old subjects, on an average, placed the reaction force the most anterior, the healthy old subjects placed it intermediately and the young subjects placed it the least anterior, within the foot support area. Moreover, the results suggest that, at liftoff, all subjects placed more importance on locating the floor reaction force to achieve acceptable postural stability than on diminishing the magnitudes of the needed joint muscle strengths.


Effects of variation on extensor elements and operative procedures in patellofemoral disorders. The influence of both insertion and strength/elasticity of each extensor in patellofemoral disorders was fully investigated through a two-dimensional mathematical model analysis in a horizontal plane, in combination with experimental design theory for analyzing mutually correlated influences. In the model, patellofemoral joint profiles projected on a horizontal plane have been expressed as spline functions. Each muscle of the quadriceps has been represented as a string pulled by the respective force; fascias and tendons have been represented by springs. Nonlinear equations have been constructed to represent the forces involved, and then solved by numerical iteration. An analysis of variance was performed on the data derived from a series of simulations, obtaining the following results. The strength of most extensors has been shown to have an influence on the increase in lateral contact force but not patellar translation. The tibial tubercle position has significant influence on both patellar translation and lateral contact force. The quadriceps' insertion on the femur has no influence on patellar translation. The insertion of each extensor on the patella has been shown to have a strong effect on patellar translation but not on contact force.


Stress distribution in a physical buttock model: effect of simulated bone geometry. Mechanical stresses developed in the tissue during sitting or reclining could cause bedsores in paralyzed individuals. Cushions are usually prescribed to redistribute the stresses. Two two-dimensional physical models of the buttock were developed and used to study whether the stress distribution is different with round- and flat-base bone core geometries and to find out whether the relative cushion responses are dependent on loading direction and bone core geometries. In these models, PVC gel simulated the soft tissue and a wooden core simulated the bony prominence. One model had a round-base core and the other had a flat-base bone core. A grid etched on the model allowed strain measurements, and stress calculations. The sharp-base bone core model generated large regions of high shear stress during vertical and inclined loading. However, the round-base core produced maximum compressive stress during vertical loading. The relative cushion responses were dependent on bone core geometry and loading direction.


A finite element model for evaluation of tibial prosthesis-bone interface in total knee replacement. A numerical model based on the finite element method was developed for the load transfer analysis at the tibial bone-implant interfaces in total knee replacement. A transverse isotropic material model, based on a quadratic elastic potential and on Hill's quadratic yield criterion, was next developed for bone constitutive laws. The bone-cement and bone-prosthesis interfaces were both assumed to be discontinuous. A dry friction model based on Coulomb's criterion was adopted for the interfaces friction. The model was shown to be able to give compressive and shear stresses distributions and distractive and relative shear micromotions at these interfaces. A preliminary application was conducted for cemented metal tray total condylar (MTTC) and for cemented and uncemented porous coated anatomic (PCA) tibial plateaus. The PCA plateaus were found to be more deformable and had greater global displacements than the MTTC one. Debonding of the bone-peg interface was observed for the uncemented PCA. Correspondingly, the stress peaks at the interface beneath the tray were lower for the uncemented PCA. Correspondingly, the stress peaks at the interface beneath the tray were lower for the PCA than for the MTTC. Shear micromotions appeared under the tray for both the two prostheses. We observed that bone anisotropy and interface discontinuity affected the results sensibly.


The behavior of adaptive bone-remodeling simulation models. The process of adaptive bone remodeling can be described mathematically and simulated in a computer model, integrated with the finite element method. In the model discussed here, cortical and trabecular bone are described as continuous materials with variable density. The remodeling rule applied to simulate the remodeling process in each element individually is, in fact, an objective function for an optimization process, relative to the external load. Its purpose is to obtain a constant, preset value for the strain energy per unit bone mass, by adapting the density. If an element in the structure cannot achieve that, it either turns to its maximal density (cortical bone) or resorbs completely. It is found that the solution obtained in generally a discontinuous patchwork. For a two-dimensional proximal femur model this patchwork shows a good resemblance with the density distribution of a real proximal femur. It is shown that the discontinuous end configuration is dictated by the nature of the differential equations describing the remodeling process. This process can be considered as a nonlinear dynamical system with many degrees of freedom, which behaves divergent relative to the objective, leading to many possible solutions. The precise solution is dependent on the parameters in the remodeling rule, the load and the initial conditions. The feedback mechanism in the process is self-enhancing, denser bone attracts more strain energy, whereby the bone becomes even more dense. It is suggested that this positive feedback of the attractor state (the strain energy field) creates order in the end configuration. In addition, the process ensures that the discontinuous end configuration is a structure with a relatively low mass, perhaps a minimal-mass structure, although this is no explicit objective in the optimization process. It is hypothesized that trabecular bone is a chaotically ordered structure which can be considered as a fractal with characteristics of optimal mechanical resistance and minimal mass, of which the actual morphology depends on the local (internal) loading characteristics, the sensor-cell density and the degree of mineralization.


Computation of steady three-dimensional flow in a model of the basilar artery. The flow in the basilar artery arises from the merging of the flows from the two vertebral arteries. To study the flow phenomena in the basilar artery, computations have been performed using a finite element (FE) method. We consider steady flow in a geometrically symmetric confluence. For simplicity, channels with a rectangular cross-section have been used. Both symmetric and asymmetric flow cases have been considered. The results show that for the Reynolds number of interest the flow downstream of the junction is highly three-dimensional, and that the flow at the end of the basilar artery, where it splits again, will not be fully developed. The computed phenomena have been confirmed by laser Doppler velocity measurements.


An activation-recruitment scheme for use in muscle modeling. The derivation of a new activation-recruitment scheme and the results of a study designed to test its validity are presented. The activation scheme utilizes input data of processed surface EMG signals, muscle composition, muscle architecture, and experimentally determined activation coefficients. In the derivation, the relationship between muscle activation and muscle fiber recruitment was considered. In the experimental study, triceps muscle force was determined for isometric elbow extension tasks varying in intensity from 10 to 100% of a maximum voluntary contraction (MVC) using both a muscle model that incorporates the activation scheme, and inverse dynamics techniques. The forces calculated using the two methods were compared statistically. The modeled triceps force was not significantly different from the experimental results determined using inverse dynamics techniques for average activation levels greater than 25% of MVC, but was significantly different for activation levels less than 25% of MVC. These results lend support for use of the activation-recruitment scheme for moderate to large activation levels, and suggest that factors in addition to fiber recruitment play a role in force regulation at lower activation levels.


Computer simulation of arterial flow with applications to arterial and aortic stenoses. A computer model for simulating pressure and flow propagation in the human arterial system is developed. The model is based on the one-dimensional flow equations and includes nonlinearities arising from geometry and material properties. Fifty-five arterial segments, representing the various major arteries, are combined to form the model of the arterial system. Particular attention is paid to the development of peripheral pressure and flow pulses under normal flow conditions and under conditions of arterial and aortic stenoses. Results show that the presence of severe arterial stenoses significantly affects the nature of the distal pressure and flow pulses. Aortic stenoses also have a profound effect on central and peripheral pressure pulse formation. Comparison with the published experimental data suggests that the model is capable of simulating arterial flow under normal flow conditions as well as conditions of stenotic obstructions in a satisfactory manner.


Effect of groove on bone fracture toughness. When testing for the effects of bone orientation on mode I fracture toughness, compact tension specimens are grooved with a V-notch to provide a crack guide. The effect of grooving on the expressions for the critical stress intensity factor (Kc) and the critical strain energy release rate (Gc) for mode I fracture toughness was investigated. Experiments were performed using grooved and ungrooved bovine compact tension specimens. The results indicate that the standard expression used to determine Kc for a compact tension specimen requires modification. The thickness (B) must be modified to account for the thickness between the grooves (Bn). The thickness used in the standard expression is replaced by an effective thickness written as (BBn)0.5. It was also found that the thickness between the grooves should be used in the standard formula for Gc.


Determination of drugs from urine by on-line immunoaffinity chromatography-high-performance liquid chromatography-mass spectrometry. A method for rapid extraction and identification of drugs in urine is described. The system utilizes a high-performance protein G immunoaffinity column coupled to a reversed-phase analytical column by use of a trapping column and switching valve. A small amount of antibody (5 micrograms drug-specific) is used for each analysis to extract either propranolol or lysergic acid diethylamide (LSD) from human urine. Urine diluted with phosphate-buffered saline is pumped directly through the protein G column thus eliminating time- and solvent-consuming sample preparation procedures. On-line ultraviolet or mass spectral analysis provides the means of drug detection and identification. With ultraviolet detection propranolol may be detected in spiked urine at the 250 pg/ml level. A Hewlett-Packard mass spectrometer modified for atmospheric pressure ionization and equipped with an ion spray source allows detection of propranolol in urine at 2.5 ng/ml and LSD at 500 pg/ml using single ion monitoring. The potential applicability of the technique for drug confirmations is discussed.


Determination of chloramphenicol in muscle using a particle beam interface for combining liquid chromatography with negative-ion chemical ionization mass spectrometry. A simple and rapid liquid chromatographic-mass spectrometric analysis of chloramphenicol in calf muscle is presented. A particle beam interface was used, with negative-ion chemical ionization mass spectrometry using methane as the reagent gas. The method specificity was tested for three related compounds, dehydrochloramphenicol, nitrosochloramphenicol and nitrophenylaminopropanediol. The extraction procedures require 5 g of muscle, and the quantification limit is 2 micrograms/kg for chloramphenicol. Residues were detected in calf muscle 48 h after intravenous administration of chloramphenicol (25 mg/kg body weight).


On-line dialysis and weak cation-exchange enrichment of dialysate. Automated high-performance liquid chromatography of pholcodine in human plasma and whole blood. An automated method for the determination of pholcodine in plasma and whole blood is described. The technique combines dialysis and trace enrichment prior to high-performance liquid chromatography. Dialysis, trace enrichment on a weak cation-exchange column, separation on a cyano column and fluorescence detection was shown to be an extremely selective and sensitive method. The method has been used successfully in the analysis of real samples after administration of pholcodine. The automated method can be used, after minor modification, to determine other basic drugs in whole blood and plasma.


Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography. A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C18 column with isocratic elution with acetate buffer (25 mM, pH 4.5)-methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35 degrees C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 microgram/ml for BA and HA and 5.0 micrograms/ml for PAA and PA with a 20-microliters injection.


High-performance liquid chromatographic determination of cyanide in human red blood cells by pre-column fluorescence derivatization. A method for the determination of cyanide in human red cells has been developed. Cyanide was extracted from red cells by adding water and methanol, and then derivatized with 2,3-naphthalene-dialdehyde and taurine to give a fluorescent product, which was determined by reversed-phase high-performance liquid chromatography with fluorescence detection. The recovery of cyanide from red cells was ca. 83%, and the limit of detection was 100 pmol/ml. The mean concentrations of red cell cyanide from ten smokers and from ten non-smokers were 705 and 466 pmol/ml, respectively. The method was also applicable to whole blood.


Rapid and sensitive determination of coumarin and 7-hydroxycoumarin and its glucuronide conjugate in urine and plasma by high-performance liquid chromatography. A rapid and sensitive high-performance liquid chromatographic method was developed for the analysis of coumarin, 7-hydroxycoumarin and its glucuronide conjugate in urine and plasma. This method was used to monitor the urinary excretion of these compounds following a single oral dose of coumarin (100 mg). This new method gives excellent chromatographic separation and includes an internal standard. The method was validated and shown to be both accurate and precise in the range 0.5-100 micrograms/ml.


High-performance liquid chromatographic determination of nicotinamide and its metabolites in human and murine plasma and urine. A high-performance liquid chromatographic method is described which enables the determination of nicotinamide and eight of its possible metabolites in human and murine plasma and urine, using ion-pairing on a base-deactivated reversed-phase column. Calibration curves were linear up to 2 mumol/ml for nicotinamide and 200 nmol/ml for the metabolites; both the intra- and inter-assay relative standard deviations ranged between 1 and 8%. In murine plasma, the N-oxide was the major nicotinamide metabolite, but in man, formation of 1-methylnicotinamide and the 2- and 4-pyridones was also significant. In urine, nicotinuric acid was seen in the mouse, but no nicotinic acid metabolites were seen in man.


Isolation and structural elucidation of the geometrical isomers of lutein and zeaxanthin in extracts from human plasma. All-E-(3R,3'R,6'R)-lutein, all-E-(3R,3'R)-zeaxanthin, all-E-(3R,3'S,6'R)-3'-epilutein and some geometrical isomers of the former two dihydroxycarotenoids have been separated from an extract of human plasma by semipreparative high-performance liquid chromatography on a silica-based nitrile-bonded column. In the order of chromatographic elution, the isolated fractions were identified as all-E-lutein, all-E-zeaxanthin, all-E-3'-epilutein, 9Z-lutein, 9'Z-lutein, a mixture of 13Z-lutein and 13'Z-lutein, 9Z-zeaxanthin, 13Z-zeaxanthin and 15Z-zeaxanthin. The structures of all compounds, including the relative configuration at C(3') and C(6') of the luteins and the position of the stereomutated double bonds in the geometrical isomers, were unambiguously established by 1H nuclear magnetic resonance spectroscopy. The absolute configuration of the three all-E compounds was derived by circular dichroism and is also assumed to be valid for the geometrical isomers. The ultraviolet-visible absorption and mass spectra of each of the individually isolated compounds were also in agreement with the proposed structures.


Determination of cocaethylene, cocaine and their metabolites in rat serum microsamples by high-performance liquid chromatography, and its application to pharmacokinetic studies in rodents. A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating cocaethylene in rat serum microsamples (50 microliters), a substance formed in vivo when cocaine and ethanol are present concurrently. The separation used a 2 mm I.D. reversed-phase Nova-Pak C18 column with a mobile phase of acetonitrile-phosphate buffer containing an ion-pairing reagent. With an ultraviolet detector operated at 230 nm, a linear response was observed from 0.05 to 2.0 micrograms/ml with a detection limit of 5 ng/ml for cocaethylene, cocaine and norcocaine. The method showed a longer half-life for cocaethylene than for cocaine in rat.


High-performance liquid chromatographic analysis of methocarbamol enantiomers in biological fluids. Methocarbamol enantiomers in rat and human plasma were quantified using a stereospecific high-performance liquid chromatographic method. Racemic methocarbamol and internal standard, (R)-(-)-flecainide, were isolated from plasma by a single-step extraction with ethyl acetate. After derivatization with the enantiomerically pure reagent (S)-(+)-1-(1-naphthyl)ethyl isocyanate, methocarbamol diastereomers and the (R)-flecainide derivative were separated on a normal-phase silica column with a mobile phase consisting of hexane-isopropanol (95:5, v/v) at a flow-rate of 1.6 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm. The resolution factor between the diastereomers was 2.1 (alpha = 1.24). An excellent linearity was observed between the methocarbamol diastereomers/internal standard derivative peak-area ratios and plasma concentrations, and the intra- and inter-day coefficients of variation were always < 9.8%. The lowest quantifiable concentration was 0.5 microgram/ml for each enantiomer (coefficients of variation of 9.8 and 8.8% for (S)- and (R)-methocarbamol, respectively), while the limit of detection (signal-to-noise ratio 3:1) was approximately 10 ng/ml. The assay was used to study the pharmacokinetics of methocarbamol enantiomers in a rat following intravenous administration of a 120 mg/kg dose of racemic methocarbamol and to evaluate plasma and urine concentrations in a human volunteer after oral administration of a 1000-mg dose of the racemate. The method is suitable for stereoselective pharmacokinetic studies in humans as well as in animal models.


Determination of acetazolamide in human urine samples by reversed-phase high-performance liquid chromatography in the presence of xanthines. A simple, rapid and selective high-performance liquid chromatographic assay for the determination of acetazolamide in urine samples is described. After extraction with ethyl acetate, the drug is chromatographed on an HP-Hypersil ODS-C18 column with a mobile phase of acetonitrile-phosphate buffer (pH 3) and ultraviolet detection at 275 nm. The efficiency of the extraction, the linearity and the reproducibility of the method permit the evaluation of acetazolamide urinary excretion a long time after its administration.


High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography. The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS-methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS-5% methanol (v/v) at 50 degrees C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.


Simultaneous measurement of serotonin, catecholamines and their metabolites in cat and human plasma by in vitro microdialysis-microbore high-performance liquid chromatography with amperometric detection. A method for the simultaneous measurement of norepinephrine, epinephrine, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin and 5-hydroxyindole-3-acetic acid in cat and human plasma by in vitro microdialysis-microbore high-performance liquid chromatography with electrochemical detection is described. The detection limit (signal-to-noise ratio = 3) is about 0.05-0.1 pg per injection. The volume of plasma samples required is very small (< 200 microliters), hence there is minimal blood loss in repeated blood sampling, especially in experiments using small animals. Within 15 min, a fast isocratic separation of these analytes by using a microbore reversed-phase ODS column is achieved, hence over 90 analyses can be performed in a single working day. As microdialysis per se is not destructive to plasma samples, the remaining plasma sample and perfusate can be repeatedly analysed for other substances. This simple, efficient and sensitive method can therefore be used as a routine clinical and basic research technique in the investigation of blood biogenic amines and their metabolites.


Quantitation of the novel anticonvulsant remacemide in rat and dog plasma and urine: application of the plasma methodology to measure the plasma protein binding of remacemide. Sensitive and selective methods have been developed for quantitation of the novel anticonvulsant remacemide in rat and dog plasma and urine. The methods employed liquid-liquid extraction (urine) or ion-exchange solid-phase extraction (plasma), with an internal standard, followed by high-performance liquid chromatography with ultraviolet detection. The detection limit for both methods was 10 ng/ml. Overall accuracy was 0.00% for plasma and -1.4% for urine with a precision of 6.04 and 3.87% for plasma and urine, respectively. The standard curves were linear for both plasma and urine over a wide concentration range (9.96-2490 ng/ml). The plasma method was also applied to measurement of in vitro plasma protein binding of remacemide in rat, dog and human plasma.


Simple and sensitive high-performance liquid chromatographic method for the determination of diltiazem and six of its metabolites in human plasma. A sensitive, specific and reproducible high-performance liquid chromatographic technique is described for the simultaneous determination in human plasma of diltiazem (DZ) and six of its primary and secondary metabolites which are products of N- and O-demethylation, deacetylation and N-oxidation. The method involves addition of excess KHCO3 to 1 ml of plasma, followed by extraction with 4 ml of ethyl acetate. The organic layer was extracted with 0.01 M HCl and the aqueous layer was dried under nitrogen and then reconstituted with 0.002 M HCl. DZ and its metabolites were free from interference and wer baseline-separated. Calibration curves were linear in the concentration range studied (5-500 ng/ml for all the species). The lower limit of quantification of the assay was 5 ng/ml for DZ and the metabolites. Inter-day and intra-day coefficients of variation were less than 10%. The applicability of this procedure is shown by evaluating the kinetics of DZ and its metabolites in three patients receiving chronic DZ therapy. N-Demethyldiltiazem, deacetyldiltiazem and N-demethyldeacetyldiltiazem were found to be the major metabolites, as previously described. Deacetyldiltiazem N-oxide was found in two of the patients. The other two known but unreported metabolites in human, O-demethyldeacetyldiltiazem and N,O-didemethyldeacetyldiltiazem, were found in the plasma of all three patients.


Determination of thalidomide in plasma and blood by high-performance liquid chromatography: avoiding hydrolytic degradation. Thalidomide was determined in plasma and blood by reversed-phase high-performance liquid chromatography with ultraviolet detection. The coefficient of variation of the assay was 1-2% over the 0.25-4.0 micrograms/ml concentration range. Hydrolysis of thalidomide during storage and work-up of the samples was avoided by the addition of an equal volume of citrate buffer, pH 1.5. The assay could be applied to the determination of blood concentrations of thalidomide in rats for at least 28 h after a single oral dose, with multiple blood sampling from the same animal.


High-performance liquid chromatographic method for quantitating plasma levels of amiloride and its analogues. An assay for amiloride was devised for efficient use with the wide variety of analogues available. Amiloride was extracted from 1-ml plasma samples by elution from a C8 preparative column with 6% acetonitrile-45% methanol-5.4% acetic acid, adjusted to pH 4.0 with trimethylamine. Samples were lyophilized, resuspended in 50% methanol, filtered through 0.22-microns Spin-X cartridges, applied to a reversed-phase C18 column, and eluted in a 0-50% acetonitrile gradient in 0.4% acetic acid, pH 4.5 (1.2 ml/min). Detection by ultraviolet absorbance at 360 nm was linear from 1 to 1000 ng. Versatility of the method was demonstrated with the analogues benzamil, 6-hydro-, 6-iodo-, 5-hexamethylene-, and 5-chlorobenzyl-2',4'-dimethylbenzyl-amiloride.


High-performance liquid chromatographic analysis of the anticancer drug oxantrazole in rat whole blood and tissues. A reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the antitumor anthrapyrazole analogue, oxantrazole (OX), in rat whole blood and tissues. Blood samples were mixed with equal volumes of a 25% (w/v) aqueous solution of L-ascorbic acid, whereas tissue samples were homogenized with 1.5-3 volumes of an L-ascorbic acid-methanol-water (1:10:1, w/v/v) mixture to prevent oxidative degradation of OX. Samples were then treated with 60% (v/v) perchloric acid (25-30 microliters/ml of stabilized sample) to precipitate proteins, and centrifuged, with the resultant supernatants analyzed on HPLC utilizing a C8 column. The mobile phase for blood and urine samples consisted of 8% (v/v) glacial acetic acid, 13% (v/v) acetonitrile, 79% (v/v) water, 0.16% (w/v) sodium acetate, and 0.05% (w/v) L-ascorbic acid (final pH 2.7), and was pumped at 1.8 ml/min. Tissue samples were eluted at 2 ml/min with a mobile phase consisting of 8% (v/v) glacial acetic acid, 12% (v/v) acetonitrile, 80% (v/v) water, 0.16% (w/v) sodium acetate, and 0.0;5% (w/v) L-ascorbic acid. OX and internal standard were detected at 514 nm and had retention times of 2.3 and 3.1 min, respectively. The limit of quantitation of OX was 25-50 ng/g. Recovery of OX from biological samples ranged from 50 +/- 0.9% in spleen to 102.8 +/- 1.8% in RG-2 glioma. The analytical method was applied to a pharmacokinetic study in rats.


Improved sample preparation for the testosterone hydroxylation assay using disposable extraction columns. The preparation of samples for injection into a high-performance liquid chromatograph from assay mixtures for the determination of cytochrome P-450-dependent testosterone hydroxylation has been substantially facilitated. By replacing the multiple cumbersome extraction steps of the conventional method with a single column extraction the time for sample preparation was reduced from hours to minutes. The new procedure also yields better recoveries for most of the testosterone metabolites than the original protocol. The use of extraction columns for sample preparation allows the simultaneous treatment of a large number of samples or even the automation of the whole assay procedure. The modified procedure is a straightforward, easy-to-perform method that should greatly facilitate the implementation of the testosterone hydroxylation assay for sharply discriminating between many individual cytochrome P-450 species in routine enzyme diagnostics.


Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection. We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C18 column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 microliters of the mobile phase. A 10-microliters aliquot of the resulting solution is injected onto a 150 mm x 4.6 mm I.D. column packed with 5-microns octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E1 = +0.25 V and E2 = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90-95% which allows the determination of 0.5 microgram/l analyte.


Ion-pair high-performance liquid chromatography of diaminopimelic acid in hydrolysates of physiological samples. A method is reported for the determination of diaminopimelic acid (DAPA) in physiological samples. DAPA is derivatized with an o-phthaldialdehyde reagent solution, subjected to reversed-phase high-performance liquid chromatography and detected spectrofluorometrically. The method is a significant advance over previous methods because it uses the ion-pairing agent hexadecyltrimethylammonium bromide (HTMA) to facilitate DAPA measurement. Ion-pairing with HTMA avoids interference with co-eluting derivatives to provide simultaneous, sensitive, reproducible measurement of both DAPA peaks (DD,LL-DAPA and DL-DAPA).