A. Jobe

Leakage of protein in the immature rabbit lung; effect of surfactant replacement

Immature newborn rabbits, delivered on day 27 of gestation, were ventilated artificially for 60 min, with or without previous treatment with natural surfactant. Insufflation pressure was adjusted to maintain an average tidal volume of about 10 ml/kg. All animals received, before the onset of ventilation, 125I-labeled albumin via the airways and 131I-labeled albumin intravenously. At the end of the experiment 3.1 +/- 1.3% (means +/- SD) of the 131I-albumin had permeated into the alveolar compartment of control animals; the corresponding figures for surfactant-treated animals were 1.7 +/- 0.8% (P less than 0.002). In control animals only 18.2 +/- 4.4% of the 125I-albumin could be recovered from the airspaces after 60 min, whereas 69.9 +/- 14.6% of this label was recovered in surfactant-treated animals (P less than 0.002). Alveolar wash samples from control animals also contained significantly increased activity of surfactant inhibitor, as evaluated with pulsating bubble. The bidirectional flux of protein, including surfactant inhibitor, was thus significantly decreased in these immature lungs by surfactant replacement.

Characterization of phospholipids and localization of some phospholipid synthetic and subcellular marker enzymes in subcellular fractions from rabbit lung

Abstract Following an alveolar wash, lung tissue from 3-day-old rabbits was gently homogenized and fractionated into organelle, cytosol, microsome and low density microsome fractions. The organelle fraction was further separated on isopycnic continuous sucrose gradients. All fractions were characterized by phosphatidylcholine and protein content, saturated-to-total phosphatidylcholine ratios, and phospholipid compositions. Rabbits were injected with radioactively labeled palmatic acid 10 min to 8 h before killing, and the specific activities (cpm/μmol) of phosphatidylcholine recovered from fractions from continuous gradients were measured. Each fraction was assayed for the presence of four subcellular marker enzymes: NADPH : cytochrome c reductase, succinate : cytochrome c reductase, 5-nucleotidase and UDPgalactose galactosyltransferase, and four phospholipid biosynthetic enzymes: cholinephosphotransferase, glycerolphosphate phosphatidyltransferase, phosphatidic acid phosphatase and lysophosphatidylcholine acyltransferase. Results were as follows: 1. Lung microsomes can be fractionated into low (under l M sucrose) and high (over 1 M sucrose) density fractions. The low density fractions have more sphingomyelin, more 5-nucleotidase and UDPgalactose galactosyltransferase activity and probably represent in part plasma membrane and Golgi fragments. 2. The organelle fraction contains a spectrum of particulate matter with phospholipids characteristic of endoplasmic reticulum fragments at high density (1.5 M sucrose) and lamellar bodies at low density (0.5 M sucrose). 3. NADPH : cytochrome c reductase was present in relatively low density fractions from the continuous gradient but not at densities characteristic of lamellar bodies, while residual 5-nucleotidase activity remained in lamellar body fractions. Cholinephosphotransferase and glycerolphosphate phosphatidyltransferase were confined to high density fractions from the continuous gradients while phosphatidic acid phosphatase and lysophosphatidylcholine acyl transferase activities were detected across the gradients. The various microsomal enzymes had disparate specific activity profiles across the continuous gradients. 4. After injection of radioactively labeled palmitic acid, radioactively labeled phosphatidylcholine sequentially enters less dense fractions with time. The distribution of the radioactively label supports the hypothesis that surfactant phospholipids move sequentially through a series of high density subcellular particles toward the low density lamellar bodies.

The Contractility and Performance of the Preterm Left Ventricle before and after Early Patent Ductus Arteriosus Occlusion in Surfactant- Treated Lambs

ABSTRACT.: The influence of left-right ductal shunting on early hemodynamic responses, namely left ventricular performance, contractility, and systemic perfusion was evaluated in nine preterm lambs (120 days gestational age) treated with surfactant. Blood gases were maintained in the physiological range using mechanical ventilation; hemodynamic and blood flow measurements (radionuclide labeled microspheres) were obtained before and after occlusion of the patent ductus arteriosus with a catheter balloon. The mean left-right ductal shunt before occlusion (1.2 h postnatal age) was 59 ± 11% SD. Left ventricular output was increased in all lambs with PDA (pre: 306 ± 106 verus post: 155 ± 31 ml/min/kg; p < 0.001); effective systemic blood flow and organ blood flows did not change. The left ventricle end-diastolic volume was increased in all and decreased following ductal occlusion (pre: 2.0 ± 0.4 verus post: 1.5 ± 0.2 ml/kg; p < 0.01). Cardiac rate, ejection fraction, and contractility (peak dP/dt) did not change. Right-left ductal shunting was not detected in six similarly treated lambs. Thus, during the 1st h of life the hemodynamic profile of preterm lambs with patent ductus arteriosus was characterized by large magnitude left-right shunt and a “high” cardiac output state sufficient to maintain unchanged systemic perfusion. The increased left ventricle output was accomplished by increasing end-diastolic volume (Frank-Starling mechanism), but left ventricle contractility remained unchanged. We speculate that the preterm left ventricle may be unable to sustain the high level of pump performance and contractility required to compensate for the ductal “steal” of systemic blood flow.

An in vivo comparison of acetate and palmitate as precursors of surfactant phosphatidylcholine

3-Day-old rabbits were injected simultaneously with [(3)H]acetate and [(14)C]-palmitic acid. The specific activities of lung, lamellar body and surfactant phosphatidylcholine and disaturated phosphatidylcholine were measured at time intervals from 10 min to 23 h following isotope administration. Palmitic acid contained 87% of the acetate radioactivity recovered from lung and surfactant phosphatidylcholine. The relative specific activities of surfactant phosphatidylcholine and disturated phosphatidylcholine labeled with acetate were 2.02 and 1.86 times those measured using the palmitic acid label. Apparently the palmitic acid synthesized from acetate is preferentially incorporated into lung phosphatidylcholines and disaturated phosphatidylcholines which are destined to become surfactant.

Clearance of large amounts of natural surfactants and liposomes of dipalmitoylphosphatidylcholine from the lungs of rabbits

Three-day-old rabbits were given intratracheal injections of radiolabeled natural sheep surfactant, rabbit surfactant, or liposomes of dipalmitoylphosphatidylcholine that contained greater than three times the quantity of phosphatidylcholine present in the endogenous surfactant pool. The recoveries of radiolabeled phosphatidylcholine and total phosphatidylcholine in alveolar washes, lung tissue, and total lung (alveolar washes plus lung tissue) were measured for 72 h. Approximately half of the two natural surfactants rapidly became lung tissue associated, and phosphatidylcholine derived from the rabbit surfactant was cleared from the total lung twice as rapidly as was the phosphatidylcholine from sheep surfactant (20.7% versus 10.4% of the amount present at zero time/24 h, p less than .05). The alveolar surfactant pool size did not decrease despite the clearance of the exogenously administered material. The liposomes of dipalmitoylphosphatidylcholine were cleared from the total lung at the same rate as the rabbit surfactant phosphatidylcholine; however, compared to the natural surfactants, much less of this material became lung tissue associated. The administration of the natural rabbit surfactant did not decrease the amount of radiolabeled choline, palmitic acid, or 32P that was incorporated into lung phosphatidylcholine or the amount of labeled phosphatidylcholine that was secreted to the alveoli. However, sheep surfactant increased the percent of radiolabeled phosphatidylcholine recovered by alveolar wash. These experiments document differences in clearance rates, tissue and alveolar association patterns, and subtle effects on endogenous surfactant metabolism for two surfactants and liposomes of dipalmitoylphosphatidylcholine following intratracheal administration. Uniform metabolic responses should not be anticipated following treatments with different surfactant preparations.

Changes in the amount of lung and airway phosphatidylcholine in 0.5–12.5-day-old rabbits

Newborn rabbits delivered spontaneously at term and cared for by the mothers were studied from 0.5 to 12.5 days of age. Curves are constructed to describe the changes in weight, lung and alveolar wash phosphatidylcholine and saturated phosphatidylcholine, and lung protein. The curves are complex and non-linear. However. expressing the increases in lung and alveolar wash phosphatidylcholine and saturated phosphatidylcholine pool sizes relative to animals weight results in a decreasing linear relationship from 0.5 to 12.5 days of age. By 12.5 days the ratios of lung phosphatidylcholine and saturated phosphatidylcholine to weight approximate the ratios measured for adult rabbits. The ratios of saturated to total phosphatidylcholine in the alveolar washes and lungs remained invarient throughout the study period.

The in vivo labeling with acetate and palmitate of lung phospholipids from developing and adult rabbits.

The labeling with radiolabeled acetate and palmitate of lung, microsomes isolated from lung, and surfactant phospholipids from adult, 3-day-old, and newborn rabbits was studied. The half-life of phosphatidylcholine from lung and microsomal fractions was shorter when labeled with acetate than when labeled with palmitate. Half-time values similarly measured for phosphatidylglycerol, phosphatidylinositol or phosphatidylethanolamine were not different for the two labels. Acetate and palmitate-labeled phospholipids appeared in the surfactant fraction with similar accumulation curves. The relative specific activities of acetate-labeled phosphatidylcholine from adult, 3-day-old, and newborn rabbits, respectively, were 1.30, 1.86 and 1.77 times those measured for those measured for the palmitate label. Surfactant phosphatidylinositol and phosphatidylethanolamine from 3-day-old animals similarly were labeled preferentially with acetate. However, phosphatidylglycerol purified from the surfactant fraction contained equivalent relative amounts of the acetate and palmitate labels in 3-day-old and adult rabbits. These results suggest that the type II pneumocyte may use acetate preferentially for the synthesis of palmitic acid which then is incorporated into surfactant phospholipids.

The Labeling of Pulmonary Surfactant Phosphatidylcholine in Newborn and Adult Sheep

The labeling of the saturated phosphatidylcholine from surfactant with radiolabeled palmitic acid was characterized in seven newborn and seven adult sheep using a repetitive sampling technique. Each animal had a small cannula placed surgically in the trachea. Following the intravenous injection of (3H) palmitic acid, surfactant samples in saline were recovered from the distal airways of each animal with fine plastic catheters over a period of 10 days. The change in specific activity of the saturated phosphatidylcholine (cpm/mumol) was used to define the kinetics of secretion and then disappearance of the labeled saturated phosphatidylcholine. Labeled saturated phosphatidylcholine accumulated in a linear fashion without an apparent initial delay for 27 hr in adult and 44 hr in newborn sheep. The labeled saturated phosphatidylcholine then decayed with mean apparent biological half-life values of 45 hr and 54 hr in adult and newborn sheep, respectively. However, these half-life estimates are compromised by the long secretory phase of the labeling curves. The characteristics of the labeling of surfactant saturated phosphatidylcholine in sheep may be more representative of surfactant metabolism in large mammals than previous studies in small rodents.

Route of incorporation of alveolar palmitate and choline into surfactant phosphatidylcholine in rabbits

Intratracheal injection of 3-day-old rabbits with radioactively labeled palmitic acid and choline results in an 8-10-fold increase in the efficiency of their incorporation into surfactant phosphatidylcholine when compared to the intravenous injection of these precursors. Based on labeling patterns in microsomal, lamellar body and alveolar wash fractions, the incorporation appears to be via normal surfactant synthetic pathways. Intratracheal injection of phospholipid precursors is useful for producing relatively high specific activity natural surfactant.

Kinetics of the in vivo labeling of the acyl groups of rabbit lung phosphatidylcholine and disaturated phosphatidylcholine

The kinetics of labeling of lung phosphatidylcholine and disaturated phosphatidylcholine were studied for periods from 0.75--120 min following intravenous injection of radiolabeled palmitic acid and choline into 3-day-old rabbits. The labeled palmitic acid was cleared rapidly from plasma, and rapidly appeared with identical incorporation kinetics in both phosphatidylcholine and disaturated phosphatidylcholine. The 2-acyl positions of both phosphatidylcholine and disaturated phosphatidylcholine were labeled preferentially soon after [14C]palmitic acid injection. The specific activities of palmitic acid in the 2-acyl positions of phosphatidylcholine and disaturated phosphatidylcholine 0.75 min after injection of labeled palmitic acid were 3.4 and 1.9 times, respectively, the specific activities of palmitic acid in the 1-acyl positions. By 120 min the label had randomized between the 1-acyl and 2-acyl positions, and the kinetics of that randomization were defined for both phosphatidylcholine and disaturated phosphatidylcholine. Choline did not pulse label lung phosphatidylcholine or disaturated phosphatidylcholine. The choline label appeared with equal specific activities in both phosphatidylcholine and disaturated phosphatidylcholine. Thus no analysis of the de novo synthesized product via the CDP-choline pathway was possible.