Machiko Ikegami

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.

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.

Size Selectivity of Lung Protein Accumulation in Preterm Ventilated Lambs

The 1-hour net accumulation of four labeled proteins of different sizes (6.5, 29, 69 and 150 kD) from the vascular space into the lungs and airspaces was measured in preterm ventilated lambs at 132 days gestational age. Lambs treated with Survanta, a surfactant prepared from bovine lung, were studied at 1, 3, 5 and 8 h after birth, while lambs not treated with this surfactant were studied up to 5 h of age because of severe respiratory failure. The labeled proteins were lost from the vascular space more rapidly over the first 1 h of life than at later times (p less than 0.01). Labeled protein recoveries were similar at 1 and 3 h in surfactant and control lambs and decreased by 8 h in surfactant-treated lambs (p less than 0.05). In both the surfactant-treated and control animals, there was a sequential decrease in labeled protein recoveries based on protein size (p less than 0.01). There was no change with time in size selectivity for accumulation of the labeled proteins into the lungs for either the control or surfactant-treated lambs, although surfactant treatments decreased accumulation of the 6.5 and 29 kD proteins at 5 h when compared to the control group (p less than 0.05). Labeled protein recoveries in alveolar washes demonstrated less size selectivity. These studies documented that size selectivity of the vascular endothelium did not change over the first 8 h of life in preterm ventilated lambs, a pattern that was not indicative of progressive lung injury.

Hemodynamic Effects of High Frequency Ventilation in Surfactant-Treated Preterm Lambs

We compared the hemodynamic status and left ventricular (LV) performance in 7 twin pairs of preterm lambs delivered at 124 days gestational age (83% of term gestation) and ventilated by either conventional ventilation (CV) or high frequency ventilation (HFV) at 15 Hz. The lambs were treated with suspensions of natural sheep surfactant to permit ventilation and survival, and ventilatory settings were adjusted to maintain physiologic blood gas values. The ductus arteriosus was occluded with a balloon catheter at 40-45 min of age to eliminate the variable of a left to right ductal shunt. Cineangiocardiographic, pressure, and blood flow measurements were made 1 and 2 h after ductal occlusion. At the same mean airway pressures, the heart rates, LV end-diastolic volumes, and mean arterial pressures were similar in both groups. LV stroke volumes, ejection fractions, LV outputs, and organ blood flows also did not differ between the two groups. When compared with CV, HFV provides comparable ventilation with no apparent deleterious hemodynamic effects in preterm surfactant-treated lambs with occluded ductus arteriosus.

Surfactant protein-B and lung function in surfactant-treated preterm lambs.

We asked if the amount of SP-B (range 37-410 micrograms/ml) in surfactants used to treat preterm lambs at 123 days of gestation correlated with postnatal lung function or the SP-B content of surfactant recovered by alveolar washes after 10 h ventilation. Ventilation was initiated using a low tidal volume strategy to minimize early lung injury. There were small increases in compliance for the lambs treated with surfactants containing more than 37 micrograms/ml SP-B and an increased lung volume for lambs treated with a surfactant containing 385 micrograms/ml/ml relative to the surfactant containing 37 micrograms/ml SP-B. The amount of SP-B in the surfactant used for treatment correlated linearly with the amount of SP-B in the surfactant recovered from the lambs (r = 0.95, p < 0.001). Albumin leak from the vasculature to the airspace was low as was total protein in alveolar washes, indicating minimal lung injury. The SP-B content of surfactant (from 37 to 410 micrograms/ml) had minimal effects on postnatal lung function over a 10-hour study period in lambs ventilated in a manner to minimize lung injury.

DISTRIBUTION OF PULMONARY BLOOD FLOW AND PROTEIN LEAK IN PREMATURE LAMBS

We studied 10 premature lambs with RDS to assess pulmonary blood flow (PBF) and protein leak distribution relative to aerated and atelectatic lung volumes. The lambs were delivered by C-section, intubated, paralyzed and ventilated so as to maintain normal blood gases. At 3.5 hrs, 125I ovalbumin (OA) was injected into an umbilical artery. At 4 hrs, radiolabeled microspheres were injected into the right ventricle to assess PBF distribution. The animals were sacrificed at 4 hrs of age, the lungs were removed, and divided into approximately 60 1 gm pieces, separating the lung into 3 categories: collapsed (C), partially aerated (P), and fully aerated (A). Each piece was assayed for 125I and microspheres. Results: 1) There was more flow to A than C regions, p<.01 in 8/10 lambs. By weight A was 36.7-70.1% (56.1%) and C was 14.9-52.1 (26.4%), while the % of total blood flow was 49.6-77.7% (64.0%) and 8.0-39.1% (19.2%), respectively. Without autoregulation there would be a mean shunt of 26.4% instead of the 19.2% measured, which could make a significant difference in the FiO2 needed to maintain normal oxygenation. Including P with C does not significantly change these results. 2) There was a relatively uniform protein leak throughout the lung but more in A than C in 4/5 animals that received OA, p<.05. 3) There was no correlation between blood flow and protein leak. Conclusions: Premature lambs with RDS have a small but significant ability for autoregulation of PBF, and intravascular protein tends to leak more in A than C regions.