Lung fluid balance in lambs before and after premature birth.

The purpose of this study was to see if lung vascular protein permeability is greater in preterm lambs with respiratory distress than it is in lambs without lung disease. We measured pulmonary vascular pressures, lung lymph flow, and concentrations of protein in lymph and plasma of 10 chronically catheterized preterm lambs (gestation 133 +/- 1 d) for 2-4 h before and for 4-8 h after delivery by cesarean section. All lambs were treated with mechanical ventilation after birth and received a constant intravenous infusion of glucose-saline solution at an hourly rate of 10 ml/kg. Respiratory failure developed in six lambs, in which there was a sustained threefold postnatal increase in lung lymph flow and lymph protein flow, with an even greater increase in pleural liquid drainage. Concentrations of protein in lymph and pleural liquid were almost identical, averaging approximately 75% of the plasma protein concentration. In the four preterm lambs without lung disease, lymph flow and lymph protein flow were either near or below fetal values by 6-8 h after birth, and there was little or no pleural liquid drainage. Extravascular lung water averaged 7.3 +/- .8 g/g dry lung in lambs with respiratory failure compared to 4.8 +/- .5 g/g dry lung in lambs without lung disease. Thus, pulmonary edema with abnormal leakage of protein-rich liquid from the lung microcirculation into the interstitium is an important pathological feature of the respiratory disease that often occurs after premature birth.

Effects of chronic alcohol consumption on the steady-state kinetics properties of cytochrome oxidase in rat liver.

The effect of chronic alcohol consumption on steady-state kinetic characteristics of cytochrome oxidase in rat liver was studied using submitochondrial particles prepared from ethanol-fed and control rats. Preparations from both control and alcoholic rats had equivalent apparent Km values for cytochrome c of 13 microM in the presence of phenazine methosulfate or 19 microM with N,N,N',N'-tetramethylphenylene diamine as oxidation-reduction mediators at physiological ionic strength. Both preparations showed comparable stimulation (approx. 3-fold) of oxidase activity following detergent solubilization of the membrane and similar temperature dependence for oxidase activity. Under all conditions, preparations from alcohol-fed rats displayed 30 to 50% lower rats of cytochrome oxidase activity per unit membrane protein than those from control rats. The diminution in specific activity per mg protein was accompanied by a similar decline in heme aa3 content, as has been noted in previous studies. When expressed on a turnover number basis, the molecular activity of cytochrome oxidase (natoms O/min per nmol heme a) was equivalent in both alcoholic and control preparations. The results indicate that the intrinsic kinetic characteristics of cytochrome oxidase are not changed by alcohol consumption. The data suggest that the characteristic decline in heme aa3 content and cytochrome oxidase specific activity seen in ethanol-fed rats does not arise from alterations in the accessibility of the oxidase towards cytochrome c, or from changes in bulk phase lipid composition or physical properties. The results support the conclusion that ethanol consumption decreases the membrane content of functionally active oxidase molecules, but does not change the catalytic properties of these oxidase molecules.

Nitric oxide decreases lung liquid production in fetal lambs.

To examine the effect of nitric oxide on fetal lung liquid production, I measured lung liquid production in fetal sheep at 130 +/- 5 days gestation (range 122-137 days) before and after intrapulmonary instillation of nitric oxide. Thirty-one studies were done in which net lung luminal liquid production (JV) was measured by plotting the change in lung luminal liquid concentration of radiolabeled albumin, an impermeant tracer that was mixed into the lung liquid at the start of each study. To see whether changes in JV might be associated with changes in pulmonary hemodynamics, pulmonary and systemic pressures were measured and left pulmonary arterial flow was measured by an ultrasonic Doppler flow probe. Variables were measured during a 1- to 2-h control period and for 4 h after a small bolus of isotonic saline saturated with nitric oxide gas (10 or 100%) was instilled into the lung liquid. Control (saline) instillations (n = 6) caused no change in any variable over 6 h. Nitric oxide instillation significantly decreased JV and increased pulmonary blood flow; these effects were sustained for 1-2 h. There was also a significant but transient decrease in pulmonary arterial pressure. Thus intrapulmonary nitric oxide causes a significant decrease in lung liquid and is associated with a decrease in pulmonary vascular resistance. In a separate series of experiments either amiloride or benzamil, which blocks Na+ transport, was mixed into the lung liquid before nitric oxide instillation; still, there was a similar reduction in lung liquid production. Thus the reduction in lung liquid secretion caused by nitric oxide does not appear to depend on apical Na+ efflux.

Pulmonary vasodilator drugs decrease lung liquid production in fetal sheep.

To examine a potential relationship between pulmonary vasodilatation and fetal lung liquid production, I measured lung liquid production in 20 fetal sheep at 130 +/- 4 days gestation while using several agents known to increase pulmonary blood flow. Thirty-two studies were done in which left pulmonary arterial flow (Qlpa) was measured by an ultrasonic Doppler flow probe and net lung luminal liquid production (Jv) was measured by plotting the change in lung luminal liquid concentration of radiolabeled albumin, an impermeant tracer that was mixed into the lung liquid at the start of each study. Qlpa and Jv were measured during a 1- to 2-h baseline period and then during a 1- to 2-h infusion period in which the fetuses received either an intravenous infusion of acetylcholine (n = 8), prostaglandin D2 (n = 10), or the leukotriene blocker FPL-55712 (n = 7). These vasodilators work by different mechanisms, each mechanism having been implicated in the decrease in pulmonary vascular resistance seen at birth. Control (saline) infusions (n = 7) caused no change in either Qlpa or Jv over 4 h. All vasodilator agents significantly increased pulmonary blood flow and decreased Jv. Pulmonary arterial pressure did not change significantly in either the control, acetylcholine, prostaglandin, or leukotriene-blocker studies, indicating that pulmonary vascular resistance decreased. Thus agents that increase pulmonary blood flow by mechanisms that occur at birth also decrease lung liquid production in fetal lambs.

Lung overexpansion increases pulmonary microvascular protein permeability in young lambs.

To study the effects of inflation pressure and tidal volume (VT) on protein permeability in the neonatal pulmonary microcirculation, we measured lung vascular pressures, blood flow, lymph flow (QL), and concentrations of protein in lymph (L) and plasma (P) of 22 chronically catheterized lambs that received mechanical ventilation at various peak inflation pressures (PIP) and VT. Nine lambs were ventilated initially with a PIP of 19 +/- 1 cmH2O and a VT of 10 +/- 1 ml/kg for 2-4 h (base line), after which we overexpanded their lungs with a PIP of 58 +/- 3 cmH2O and a VT of 48 +/- 4 ml/kg for 4-8 h. QL increased from 2.1 +/- 0.4 to 13.9 +/- 5.0 ml/h. L/P did not change, but the ratio of albumin to globulin in lymph relative to the same ratio in plasma decreased, indicating altered protein sieving in the pulmonary microcirculation. Seven other lambs were mechanically ventilated for 2-4 h at a PIP of 34 +/- 1 cmH2O and a VT of 23 +/- 2 ml/kg (base line), after which their chest and abdomen were bound so that PIP increased to 54 +/- 1 cmH2O for 4-6 h without a change in VT. QL decreased on average from 2.8 +/- 0.6 to 1.9 +/- 0.3 ml/h (P = 0.08), and L/P was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased pulmonary vascular filtration pressure does not alter lung liquid secretion in fetal sheep.

The purpose of this study was to determine whether an increase in pulmonary vascular filtration pressure affects net production of liquid within the lumen of the fetal lung. We studied 14 chronically catheterized fetal lambs [130 +/- 3 (SD) days gestation] before, during, and after a 4-h rapid (500 ml/h) intravenous infusion of isotonic saline. In seven fetuses we measured pulmonary arterial and left atrial pressures, lung lymph flow, and protein osmotic pressures in plasma and lymph. In eight lambs with a chronically implanted tracheal loop cannula, we measured the change in luminal lung liquid volume over time by progressive dilution of tracheally instilled 125I-albumin, which stays within the lung lumen. Saline infusion increased pulmonary vascular pressures by 2-3 mmHg and decreased the plasma-lymph difference in protein osmotic pressure by 1 mmHg. Lung lymph flow increased from 1.9 +/- 0.6 to 3.9 +/- 1.2 (SD) ml/h; net production of luminal lung liquid did not change (12 +/- 5 to 12 +/- 6 ml/h). Thus an increase in net fluid filtration pressure in the pulmonary circulation, which was sufficient to double lung lymph flow, had no significant effect on luminal lung liquid secretion in fetal sheep.

Lung vascular protein permeability in preterm fetal and mature newborn sheep.

The purpose of this study was to see whether there are developmental differences in the protein permeability of the pulmonary circulation that might contribute to the abnormal lung fluid balance seen in premature lambs with respiratory failure. In one series of experiments, we measured albumin turnover time, which reflects the escape rate of radiolabeled albumin from the pulmonary circulation, of five preterm fetal lambs (125 +/- 1 days gestation) and five newborn lambs (19 +/- 9 days old). Turnover time was not significantly different in fetuses (160 +/- 38 min) and newborns (141 +/- 54 min), implying a similar protein permeability of the pulmonary circulation. In additional experiments, we measured pulmonary hemodynamic and lung lymph flow responses to intravenous saline infusion in seven preterm fetal lambs (130 +/- 3 days gestation) and seven newborn lambs (14 +/- 3 days old). During saline infusion, calculated fluid filtration pressure increased by a similar amount in fetuses and newborns (3.4 +/- 0.8 and 2.8 +/- 0.9 Torr, respectively), resulting in a similar change in lung lymph flow in fetuses and newborns (0.59 +/- 0.27 and 0.55 +/- 0.25 ml.h-1.kg body wt-1, respectively). The results of these studies indicate that protein permeability of the pulmonary circulation does not change significantly during late fetal and early postnatal development.

Hypoproteinemia slows lung liquid clearance in young lambs.

To determine whether hypoproteinemia slows the rate at which liquid is cleared from the lung lumen, we studied 36 lambs, 18 of which underwent repeated plasmapheresis, reducing plasma protein concentration by 37% and plasma protein osmotic pressure by 39%. We killed 29 lambs (14 hypoproteinemic and 15 normoproteinemic) and removed their lungs 1, 2, or 6 h after intratracheal instillation of isotonic saline (6 ml/kg body wt). We measured extravascular lung water and determined the percentage of tracheally instilled liquid that was cleared from the lungs by comparison with control lambs that did not receive saline into their airways. The percent liquid cleared from the lungs after 1 and 2 h was significantly less in hypoproteinemic than in normoproteinemic lambs (37 vs. 65% at 1 h, 58 vs. 75% at 2 h, respectively). By 6 h nearly all the liquid (> 92%) was cleared from the lungs of all lambs. Thus hypoproteinemia slows the initial rate of clearance of liquid from the lungs of lambs. To determine whether reduced plasma protein osmotic pressure might redirect this liquid into lung lymphatics, we measured lung lymph flow (Q1) in five lambs (7.7 +/- 1.4 kg, 19 +/- 4 days old) for > or = 2 h before and 6 h after tracheal instillation of saline. In each lamb, paired studies were done 3-6 days apart; between studies the lambs underwent plasmapheresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in lung lipid during spontaneous labor in fetal sheep.

The goals of this study were 1) to examine changes in lung liquid formation and composition during spontaneous labor in fetal lambs and 2) to determine the importance of beta-adrenergic stimulation and transepithelial Na+ flux in removing liquid from the lung lumen near birth. We measured net production of lung liquid (Jv), lung liquid composition, and transpulmonary electrical potential difference (PD) before and during labor in fetal sheep with chronically implanted tracheal and vascular catheters. We determined Jv by measuring rate of change in lung liquid concentration of 125I-albumin, an impermeant tracer that was mixed in lung liquid at the start of each study. In 17 paired experiments, Jv decreased from 11 +/- 2 ml/h (Jv > 0 = secretion) before labor to -1 +/- 2 ml/h (Jv < 0 = absorption) during labor; in 5 paired experiments, PD changed from -7 +/- 1 mV (lumen negative) before labor to -12 +/- 1 mV during labor. To determine whether absorption of lung liquid during labor is the result of beta-adrenergic stimulation, we studied the effect of propranolol on Jv during labor. When propranolol (40 microM) was added to lung liquid during active labor, Jv decreased from -2 +/- 2 to -8 +/- 3 ml/h (n = 9). Thus, propranolol did not inhibit lung liquid absorption during labor. To determine whether transepithelial Na+ movement provides the driving force for lung liquid clearance during labor, we tested the effects of amiloride, an Na+ transport inhibitor, on Jv and PD.(ABSTRACT TRUNCATED AT 250 WORDS)

Pre- versus post-ventilatory surfactant treatment in surfactant-deficient preterm lambs.

Twenty lambs at 127 days' gestation (term is 145 days) were randomly assigned to receive Infasurf (Calf Lung Surfactant Extract, ONY Inc., Amherst, NY) as an intratracheal bolus (3 mliter kg-1) either into a fluid-filled lung before ventilation (n = 10), or after ventilation for 5 min (n = 10). All lambs were surfactant-deficient by analysis of lung liquid obtained before surfactant administration. Lambs were then mechanically ventilated for 4 h. Oxygenation for the lambs given surfactant before ventilation did not change during the experiment; a/A pO2 was 0.50 +/- 0.13 at 1 h and 0.52 +/- 0.17 at 4 h. For the lambs given surfactant after initial ventilation, oxygenation decreased over time; a/A pO2 decreased from 0.48 +/- 0.23 at 1 h to 0.37 +/- 0.22 at 4 h (P < 0.05). Compliance, as calculated from the Ventilator Efficiency Index (VEI), improved over time in both groups, but was always significantly higher for lambs given surfactant before ventilation (P = 0.03). Histologic examination of the lungs revealed no differences between the groups; no evidence of epithelial denudation or hyaline membrane formation was seen in either group. Thus, ventilation of surfactant-deficient newborn lambs for 5 min before surfactant administration results in significantly decreased lung function when compared with surfactant administration before ventilation. These differences in lung function are not dependent on a histopathologic injury to the lung. It is possible that unevenness of deposition of the surfactant in an air-filled lung, compared to more uniform deposition in a fluid-filled unventilated lung, produces these differences.

Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times?

Peptide mapping is a key analytical method for studying the primary structure of proteins. The sensitivity of the peptide map to even the smallest change in the covalent structure of the protein makes it a valuable 'finger-print' for identity testing and process monitoring. We recently conducted a full method validation study of an optimised reverse-phase high-performance liquid chromatography (RP-HPLC) tryptic map of a therapeutic anti-CD4 IgG1 monoclonal antibody. We have used this method routinely for over 1 year to support bioprocess development and test production lots for clinical trials. Herein we summarize the precision and ruggedness of the testing procedure and the main findings with respect to 'coverage of amino acid sequence' and limits-of-detection for various hypothetical structural variants. We also describe, in more detail, two unanticipated insights into the method gained from the validation study. The first of these is a potentially troublesome side-product arising during the reduction/alkylation step. Once the cause of this side-product was identified, it was easily prevented. We also report on subtle changes to the peptide map upon extended storage of the digest in the autosampler. These findings helped us to develop a 'robust' method for implementation in a quality control laboratory.

Nitric oxide decreases lung liquid production via guanosine 3',5'-cyclic monophosphate.

We studied the role of cGMP in nitric oxide (NO)-induced changes in lung liquid production (J(v)) in chronically instrumented fetal sheep. Forty-five studies were done in which J(v) was measured by a tracer dilution technique. Left pulmonary arterial flow (Q(lpa)) was measured by a Doppler flow probe. There were two series of experiments. In the first, we gave 8-bromo-cGMP, a cGMP analog, by either the pulmonary vascular or intraluminal route; in the second, we used agents to inhibit or enhance endogenous cGMP activity. When infused directly into the pulmonary circulation, 8-bromo-cGMP significantly increased Q(lpa) but had no effect on J(v). Conversely, when instilled into the lung liquid, 8-bromo-cGMP had no effect on Q(lpa) but significantly reduced J(v). Inhibition of guanylate cyclase activity with methylene blue totally blocked, whereas phosphodiesterase inhibition with Zaprinast significantly enhanced, the effect of instilled NO on J(v). Thus the reduction in lung liquid caused by NO appears to be mediated by cGMP, perhaps through a direct effect on the pulmonary epithelium.

Acetylcholine increases pulmonary blood flow in intact fetuses via endothelium-dependent vasodilation.

In vitro, acetylcholine causes vasodilation by releasing endothelium-derived relaxing factor (EDRF) from endothelial cells. EDRF may be nitric oxide, derived from the amino acid L-arginine (L-Arg), by a process that is inhibited by NG-monomethyl-L-arginine (L-NMMA) and restored by L-Arg. We studied the effect of L-NMMA and L-Arg on the increase in pulmonary blood flow caused by acetylcholine in unanesthetized intrauterine near-term fetal lambs. Three protocols were employed. In each protocol, acetylcholine (0.48 +/- 0.15 micrograms/kg) was injected at 15-min intervals for 120 min. In the control protocol, nothing else was given. In the second protocol, L-NMMA (14 +/- 5 mg/kg) was given at 35 min. In the third protocol, L-NMMA was given at 35 min followed by L-Arg (138 +/- 73 mg/kg) at 80 min. In the control protocol, acetylcholine increased pulmonary blood flow 179 +/- 17% while it decreased pulmonary arterial pressure 15 +/- 1% and did not affect left atrial pressure. The response to each injection lasted less than 1 min and did not change throughout the experiment. L-NMMA completely blocked, whereas L-Arg completely restored, the effect of acetylcholine on pulmonary blood flow. We conclude that acetylcholine increases pulmonary blood flow in the fetal lamb via the release of EDRF derived from L-Arg. We speculate that endothelium-dependent vasodilation may play a role in the increase in pulmonary blood flow at birth.

A controlled trial of dexamethasone in preterm infants at high risk for bronchopulmonary dysplasia.

We evaluated the use of dexamethasone in preterm infants to decrease morbidity associated with bronchopulmonary dysplasia in a randomized, double-blind, placebo-controlled trial. Thirty-six preterm infants (birth weight, less than or equal to 1250 g and gestational age, less than or equal to 30 weeks) who were dependent on oxygen and mechanical ventilation at two weeks of age received a 42-day course of dexamethasone (n = 13), an 18-day course of dexamethasone (n = 12), or saline placebo (n = 11). The starting dose of dexamethasone was 0.5 mg per kilogram of body weight per day, and it was progressively lowered during the period of administration. Infants in the 42-day dexamethasone group, but not those in the 18-day group, were weaned from mechanical ventilation significantly faster than control infants (medians 29, 73, and 84 days, respectively; P less than 0.05), and from supplemental oxygen (medians 65, 190, and 136 days, respectively; P less than 0.05). No clinical complications of steroid administration were noted. Follow-up of all 23 survivors at 6 and 15 months of age showed good outcome (normal neurologic examinations and Bayley Developmental Indexes greater than or equal to 84) in 7 of the 9 infants in the 42-day dexamethasone group, but in only 2 of the 9 infants in the 18-day dexamethasone group and 2 of the 5 in the placebo group (P less than 0.05). We conclude that dexamethasone therapy for 42 days improves pulmonary and neurodevelopmental outcome in very-low-birth-weight infants at high risk for bronchopulmonary dysplasia.

Ion transport regulation of lung liquid secretion in foetal lambs.

To test the hypothesis that liquid formation in the foetal lung reflects the balance between Cl- secretion and Na+ absorption by the respiratory tract epithelium, we studied the independent and combined effects of selective ion transport inhibitors on basal production of lung liquid in foetal lambs. We prepared 19 foetal lambs (gestation 125 +/- 4, term = 147 days) with chronic indwelling catheters for subsequent measurement of luminal liquid production over time (JV). Using an impermeant tracer technique, we measured JV before and after tracheal instillation of 2 different inhibitors of ion transport: bumetanide, a Na(+)-K(+)-2Cl- co-transport inhibitor, and amiloride, a Na+ transport inhibitor. In 7 foetuses we sequentially added bumetanide (10(-4) M) and 2 different concentrations of amiloride (10(-6) M, 10(-4) M) to the liquid within the lung lumen. After we gave bumetanide, JV decreased from 12 +/- 4 ml/h to 0 +/- 5 ml/h and subsequently increased during the 2 periods of amiloride exposure (10(-6) M: 6 +/- 5 ml/h; 10(-4) M: 7 +/- 7 ml/h). In 5 control studies we gave bumetanide, followed by only amiloride vehicle. JV for all time periods in the control studies was similar to the experimental group, demonstrating no effect of amiloride. In 5 foetuses we administered the 2 concentrations of amiloride before bumetanide. There was no change in JV with either concentration of amiloride (baseline: 13 +/- 2 ml/h; 10(-6) M amiloride: 15 +/- 5 ml/h; 10(-4) M amiloride: 13 +/- 6 ml/h).(ABSTRACT TRUNCATED AT 250 WORDS)