Androgen regulation of lung lipids in the male rat

The aim of this study was to determine whether or not testosterone regulates the lipid concentration in rat lung tissue. Rats were either sham-operated controls, castrated, or castrated and injected with testosterone. Twenty-one days after castration, we observed in relation to the control: (i) Total lipids, phospholipids, and total cholesterol increased, while triglycerides decreased in whole lung. (ii) Phospholipid concentration increased in microsomes, lamellar bodies, and alveolar macrophages, but it decreased in extracellular surfactant. (iii) On a percentage basis, the concentration of phosphatidylcholine increased in microsomes, lamellar bodies, and alveolar macrophages, and it decreased in extracellular surfactant. (iv) Protein concentration decreased in extracellular surfactant and increased in microsomes, lamellar bodies, and alveolar macrophages. (v) The incorporation of [¹⁴C]glycerol into phospholipids of lung slices increased. (vi) The activity of CTP:phosphocholine cytidylyltransferase bound to the microsomal fraction increased without any change in the activity of the soluble form of the enzyme in the lung. The results obtained when testosterone was administered to castrated rats were similar to those obtained in the control in all cases. These results suggest that the lipid concentration in the lung is regulated at least partly directly or indirectly by androgens.     

Turnover of cholesterol and dipalmitoyl phosphatidylcholine in surfactant of adult rat lung

In order to compare the turnover of two major surfactant components, [1α,2α(n)-³H]cholesterol and [methyl ¹⁴ C choline] dipalmitoyl phosphatidylcholine (DPPC) were introduced as lamellar bodiesvia the trachea into lightly anesthetized rats which were then allowed to recover. The radiotracers were assumed to have entered the alveolar surfactant pool and to have subsequently recycled in part into the lamellar bodies of alveolar type II cells. For DPPC, the specific activityvs. time curves of tubular myelin rich (alv-1) and tubular myelin poor (alv-2) alveolar lavage fractions were similar, and there was a plausible precursor-product relationship between lamellar bodies and either (or both) of these compartments. In contrast, however, the specific activities of alv-1 and alv-2 for cholesterol were quite different, allowing us to reject the hypothesis of a precursor-product relationship between classical lamellar bodies and alv-2. The estimated turover time for DPPC in alv-1 was 240 or 206 min, depending on which subfraction of lamellar bodies one takes to be the precursor. For cholesterol it was 583 or 624 min. These longer turnover times for cholesterol should lead to a greater than twofold increase in the relative concentration of cholesterol in the putative product compartment. Such an increase was not found. We interpret this as reflecting either noncompartmental behavior of the alveolar surfactant pool, or multiple pools of lamellar bodies with different turnover times. We conclude that two major components of pulmonary surfactant, cholesterol and DPPC, are handled differently, and that for at least one of these substances, the widely accepted scenario of a compartmental precursor-product relationship between lamellar bodies and alveolar surfactant must be rejected.     

Coordinate packaging of newly synthesized phosphatidylcholine and phosphatidylglycerol in lamellar bodies in alveolar type II cells

Methylamine, a weak base, inhibits packaging of newly synthesized phosphatidylcholine (PC) in lamellar bodies in 20–22 h cultured alveolar type II cells, suggesting a role for acidic pH of lamellar bodies. In this study, we tested if (i) the packaging of PC is similarly regulated in freshly isolated type II cells and (ii) methylamine also inhibits the packaging of other surfactant phospholipids, particularly, phosphatidylglycerol (PG). The latter would suggest coordinated packaging so as to maintain the phospholipid composition of lung surfactant. During the short-term metabolic labeling experiments in freshly isolated type II cells, methylamine treatment decreased the incorporation of radioactive precursors into PC, disaturated PC (DSPC), and PG of lamellar bodies but not of the microsomes, when compared with controls. The calculated packaging (the percentage of microsomal lipid packaged in lamellar bodies) of each phospholipid was similarly decreased (∼50%) in methylamine-treated cells, suggesting coordinated packaging of surfactant phospholipids in lamellar bodies. Equilibrium-labeling studies with freshly isolated type II cells (as is routinely done for studies on surfactant secretion) ± methylamine showed that in methylamine-treated cells, the secretion of PC and PG was decreased (possibly due to decreased packaging), but the phospholipid composition of released surfactant (measured by radioactivity distribution) was unchanged; and the PC content (measured by mass or radioactivity) of lamellar bodies was lower, but the PC composition (as percentage of total phospholipids) was unchanged when compared with control cells. We speculate that the newly synthesized surfactant phospholipids, PC, DSPC, and PG, are coordinately transported into lamellar bodies by a mechanism requiring the acidic pH, presumably, of lamellar bodies.     

Source of lung surfactant phospholipids: Comparison of palmitate and acetate as precursors

The phospholipids and the fatty acid compositions of major phospholipids in rat lung parenchyma, microsomes, lamellar bodies and alveolar wash were quantified. Adult rats were injected simultaneously with [³H] palmitate and [¹⁴C] acetate into the femoral vein. The appearance of labeled phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC) and phosphatidylglycerol (PG) in each lung fraction was measured during short periods of time (5 min to 2 hr) after isotope administration. Relatively more PC, DSPC and PG labeled with acetate radioactivity in lung microsomes entered lamellar body and alveolar wash fractions than those labeled with palmitate radioactivity. However, there was no difference between palmitate and acetate labeled phospholipids in the transport from microsomes to lamellar bodies by phospholipid exchange proteins. On the other hand, prior injection of colchicine resulted in decrease in the transport of PC from microsomes to alveolar space to a relatively greater extent in the acetate radioactivity than in the palmitate radioactivity.     

Analysis of subcellular phosphatidyl choline in developing rabbit lung

Phosphatidyl choline is a major lung surfactant. Insufficient development of the surfactant in neonates is often associated with the Respiratory Distress Syndrome. The concentration and fatty acid composition of phosphatidyl choline have not been studied in the subcellular organelles of the developing lung. This study has investigated the development of the concentration and fatty acid composition of phosphatidyl choline in subcellular fractions of 28-day and 30-day fetal and maternal New Zealand rabbit lungs. The concentration of total phospholipids in lamellar bodies increased four to five fold from 28-day fetus to 30-day fetus which, in turn, was similar to the maternal level. Total phospholipid content increased only about 50% in mitochondria and microsomes. The percentage of phosphatidyl choline among total phospholipids in lamellar bodies increased successively from 60% at 28 days gestation to 84% at 30 days gestation and leveled at 84% in maternal lamellar bodies. Microsomal PC increased steadily from 52% in the 28-day fetus to 65% in the adult. Analysis of the fatty acid composition of phosphatidyl choline in lamellar bodies confirmed 16∶0 as the major fatty acid, and its content remained constant from 28 days gestation to adult. In contrast, the content of 16∶0 of the microsomal phosphatidyl choline decreased with increasing gestation. Changes of several unsaturated fatty acid components were observed in both lamellar bodies and microsomes in the developing lungs. Maturational development of phosphatidyl choline is reflected in an increase in the concentration of this surfactant, particularly in lamellar bodies, and possibly in remodeling of fatty acid composition in both lamellar bodies and microsomes.     

Subcellular distribution of disaturated phosphatidylcholine in developing rabbit lung

To determine the subcellular distribution of disaturated phosphatidylcholine (DSPC) in lung tissue during perinatal development, fetal rabbits at 24, 26, 28 and 31 (term) days gestation and newborns were studied. Following alveolar lavage, fractions enriched in nuclei-cellular debris, mitochondria, microsomes, surfactant (lamellar bodies) and cytosol were prepared from the residual tissue homogenate, and their DSPC content was determined. The DSPC content of the unfractionated residual lung tissue homogenate progressively and significantly increased during fetal development, rising from 9.09±0.91 to 17.45±2.88 mg/g dry lung between 24 days gestation, and term. Between 24 and 26 days gestation the overall increase in tissue DSPC was due to a two-fold increase in the mitochondrial, microsomal and cytosolic pools. Lamellar bodies were first isolable at 26 days gestation. The DSPC content of this fraction increased six-fold (from 0.10±0.02 to 0.67±0.15 mg/g dry lung) between 26 and 28 days gestation and a further seven-fold (to 4.63±1.06 mg/g dry lung) by term, accounting for the overall increase in the tissue homogenate value during this time period. By the first postnatal day, microsomal and cytosolic DSPC increased another two-fold, but no significant change occurred in the other subcellular fractions. Alveolar lavage DSPC progressively increased over the time period studied. While there was no change in the lamellar body DSPC/total PC ratio during fetal development, each of the mitochondrial, microsomal and cytosolic ratios decreased between days 26 and 28 of gestation and then increased at term. Our results indicate that in addition to the pulmonary surfactant, for which DSPC is often used as a marker, other subcellular organelles contain significant DSPC pools that undergo dynamic changes in size during perinatal development.     

Vitamin E-Stoffwechsel der Lunge

Vitamin E Metabolism of the Lung . Experimental and clinical data have provided evidence for the involvement of oxygen free radicals in the development of acute and chronic lung diseases. Alveolar surfactant is the prime target of oxidative air pollutants. Lung surfactant consists to 90% of lipids and contains vitamin E as most important lipophilic antioxidant. Recently, we showed that alveolar surfactant is supplemented with vitamin E during its synthesis in type II pneumocytes. Hyperoxia is very often a necessary therapeutic intervention which seems to impose oxidative stress on lung tissue. Hyperoxia caused an increase in vitamin E turnover, measured in type II pneumocytes, lamellar bodies and lung lavages. In contrast, the turnover of surfactant cholesterol and surfactant lipids does not change. Hyperoxia caused an increase in vitamin E uptake by type II pneumocytes an enrichment of vitamin E in lamellar bodies.     

Lung lipid composition in zinc-deficient rats

There have been a limited number of studies investigating surfactant lipid changes in lung with trace elements. The present investigation was designed to examine the effect of moderate zinc deficiency on the lipid metabolism in rat lung. We also evaluated whether zinc deficiency, which is a wide-spread problem, could play a role in adult respiratory distress syndrome (ARDS). For that purpose, adult male Wistar rats were fed two diets differing in zinc concentration. The rats were divided into two groups. One group was fed a zinc-deficient diet containing 3 mg Zn/kg, and the other group received a zincadequate control diet with 30 mg Zn/kg according to AIN 93-M. After 2 mon of treatment, we observed that in the zinc-deficient group (i) total lipids, phospholipids, and cholesterol increased whereas TG decreased in whole lung; (ii) phospholipid (PC) concentration increased in lamellar bodies and alveolar macrophages and decreased in extracellular surfactant but did not change in microsomes; (iii) protein concentration decreased in whole lung, extracellular surfactant, lamellar bodies, and macrophages; (iv) the incorporation of [Me-¹⁴C]choline into PC (phospholipids) of lung slices increased; and (v) the activity of CTP/phosphocholine cytidylyltransferase bound to the microsomes increased in the lung. These results suggest that the lipid concentration in the lung (especially the phospholipids) is modified directly or indirectly by a zinc-deficient diet. In a zincdeficient diet, the lung changes the pattern of PC for an adaptive or recovery stage. Therefore, zinc deficiency implications are important for the design of therapies and public health interventions involving targeted zinc supplementation for high-risk groups or groups with certain diseases, such as ARDS.     

The phospholipids of rabbit type II alveolar epithelial cells: Comparison with lung lavage,lung tissue,alveolar macrophages,and a human alveolar tumor cell line

The phospholipid composition of type II alveolar epithelial cells from the rabbit was compared with that of alveolar macrophages, lung lavage and lung tissue. In addition, the phospholipid composition of a human alveolar tumor cell line, which is morphologically similar to type II cells, was examined. Phosphatidylcholine accounted for 48% of the total phospholipid in the type II cells, 41% in the tumor cells, and 30% in the macrophages. Phosphatidylcholine was 51% disaturated in the type II cells, 54% in lung lavage, 39% in whole lung, 29% in lavaged lung and macrophages, and 16% in the tumor cells. Palmitic acid was the major fatty acid in phosphatidylcholine from all samples with the exception of the tumor cells in which almost half of the fatty acids were accounted for by oleic acid. The phospholipids of the type II cells were more similar to those of lung lavage, and thus surfactant, than to lung tissue and macrophages. This is consistent with their supposed role in surfactant production. The tumor cells, although morphologically similar to type II cells, were quite different with respect to phospholipid composition.     

Fatty Acid Profile Analysis of Membrane Phospholipids of Isolated Soybean Lipid Bodies

Phospholipids in isolated soybean lipid bodies at 30‡C underwent degradation by acyl ester and phosphodiester hydrolysis and by phosphatidyl transfer. Under conditions that minimized oxidation, preferential loss of phosphatidylethanolamine and polyunsaturated fatty acids occurred, but acyl ester compositions of all phospholipids reflected enrichment of saturated and/or monounsaturated acids. Such fatty acid changes in phosphatidylinositol, which was degraded less than phosphatidylcholine or phosphatidylethanolamine, and in phosphatidic acid, which accumulated, also suggest that transesterification occurred extensively in lipid bodies. The specific temporal changes in phospholipid and acyl chain composition suggest that several enzymes remained active in isolated lipid bodies.     

Effect of hyperpnea on enzymes of the CDPcholine path for phosphatidylcholine synthesis in rat lung

We have examined the activity of three enzymes in pulmonary surfactant phosphatidylcholine synthesis following the hyperpnea induced by having rats either inspire 5% CO₂/13%O₂/82% N₂ for 24 hr or swim in thermoneutral water for 30 min. Both stimuli markedly increase frequency and tidal volume of breathing and promote the release of surfactant. Lungs were perfused to remove blood, lavaged, and then homogenized in 1 mM Hepes, 0.15M KCl at pH 7.0. The homogenate was centrifuged at 9,000 g (av) for 10 min to sediment the mitochondria and lamellar bodies and at 100,000 g (av) for 60 min to obtain the microsomal and cytosol fractions. Incubations were carried out under determined optimal conditions and zero order kinetics. Choline kinase (CK), cholinephosphate cytidylytransferase (CP-cyT) and choline phosphotransferase (CPT) were assayed by the incorporation of [methyl-¹⁴C] choline chloride into phosphocholine, [methyl-¹⁴C]phosphocholine into CDPcholine, and [¹⁴C]CDPcholine into phosphatidylcholine, respectively. The incubation products were separated by thin-layer chromatography. Whereas both forms of hyperpnea increased the activity of CP-cyT in the microsomal fraction, they had no effect on the activity of either cytosolic CP-cyT and CK, or microsomal CPT. A similar increase in tidal volume in an isolated perfused rat lung had no effect. We conclude that,in vivo, hyperpnea increases the activity of CP-cyT, the rate-limiting enzyme in phosphatidylcholine synthesis. Whether this is due to an increase in the amount of enzyme, or of a cofactor, is unknown.     

Ozonation at Wiggins Water Purification Works,Durban, South Africa

The post-ozone plant at Wiggins waterworks was commissioned during 1984. The main objective of the post–ozone plant was to ensure proper disinfection, including the elimination of viruses, whenever raw water for the works was pumped from the polluted lower Umgeni River. The main problems experienced with the post–ozone plant were poor ozone transfer efficiency in the contact tank and poisoning of the catalyst in the thermal-catalytic destructor.     

Lung lamellar bodies lack certain key enzymes of phospholipid metabolism

Palmitoyl CoA-glycerol-3-phosphate acyltransferase, phosphatidate phosphohydrolase, and phospholipase A were assayed in subcellular fractions of rat lung, including lamellar bodies, the putative site of storage and secretion of lung surfactant. The specific activity of each of these enzymes in lamellar bodies was relatively low and could be entirely accounted for by a small contamination of the lamellar bodies fraction by microsomes, as quantitated by the presence of the microsomal marker reduced triphosphopyridine nucleotide cytochromec reductase. These data indicate that lamellar bodies are not the site of synthesis of the lipid component of pulmonary surfactant by pathways involving these enzymes.     

Mouse alveolar surfactant: Characterization of subtypes prepared by differential centrifugation

To characterize the properties of alveolar surfactant susbfractions obtained from mouse lung by differential centrifugation, lavage fluid, following a preliminary centrifugation at 140 × g for 5 min to yield a cellular pellet (Pc), was sequentially cetrifuged at 10,000 ×g for 30 min, 60,000 ×g for 60 min and 100,000 ×g for 15 h; and the resultant pellets, respectively referred to as P10, P60 and P100, were harvested for electron microscopy, phospholipid analysis and surface tension measurements. Ultrastructural differences were observed, in that P10 contained large multilamellated structures which were typical of newly secreted surfactant, P100 contained small unilamellar vesicular structures, typical of catabolic end products of alveolar surfactant and P60 appeared to contain a mixture of structures present in P10 and P100 in addition to numerous, large unilamellar vesicles which were not present in either P10 or P100. Slight but significant differences were found in the phospholipid compositions of the three subfractions but not in the fatty acid composition of their phosphatidylcholine (PC) component. There were no significant differences in their disaturated PC/total PC ratios, but significant differences in their phospholipid/protein ratios. P60 had the highest proportion of phospholipid to protein. P10 and P60 demonstrated surface activity but P100 did not. Total alveolar surfactant phospholipid was evenly distributed among the three fractions. This pattern of distribution was significantly different from that observed in rabbit subfractions prepared by the same procedure. These data indicate that mouse alveolar surfactant consists of three distinct subfractions or subtypes which can be separately and quantitatively isolated by differential centrifugation. They also suggest that there may be species differences in the relative proportions of the individual subtypes present in normal adult lung.     

Myelin and Lipid Composition of the Corpus Callosum in Mucopolysaccharidosis Type I Mice

Mucopolysaccharidosis type I (MPS I) is a lysosomal disease with progressive central nervous system involvement. This study examined the lipid, cholesterol, and myelin basic protein composition of white matter in the corpus callosum of MPS I mice. We studied 50 week-old, male MPS I mice and littermate, heterozygote controls (n = 12 per group). Male MPS I mice showed lower phosphatidylcholine and ether-linked phosphatidylcholine quantities than controls (p < 0.05). Twenty-two phospholipid or ceramide species showed significant differences in percent of total. Regarding specific lipid species, MPS I mice exhibited lower quantities of sphingomyelin 18:1, phosphatidylserine 38:3, and hexosylceramide d18:1(22:1) mH₂O than controls. Principal components analyses of polar, ceramide, and hexosylceramide lipids, respectively, showed some separation of MPS I and control mice. We found no significant differences in myelin gene expression, myelin basic protein, or total cholesterol in the MPS I mice versus heterozygous controls. There was a trend toward lower proteolipid protein-1 levels in MPS I mice (p = 0.06). MPS I mice show subtle changes in white matter composition, with an unknown impact on pathogenesis in this model.     

Effects of ethanol diets on cholesterol content and phospholipid acyl composition of rat hepatocytes

Chronic treatment of adult male rats with ethanol liquid diets resulted in alterations in phospholipid and cholesterol contents as well as the acyl composition of phosphatidylethanolamine (PE), phosphatidylinositol (PI)-phosphatidylserine (PS) mixture, and phosphatidylcholine (PC) of isolated hepatocytes. The influence of ethanol on these lipids was largely dependent on the proportion of dietary fat. Phospholipid and total cholesterol contents were elevated 23 and 27%, respectively, by ethanol when offered in a low-fat diet (5% corn oil). Only the percentage of arachidonic acid from PI-PS was significantly reduced in the low-fat ethanol group. Exposure to a high-fat (34% corn oil) diet in the presence of ethanol for 4–5 weeks resulted in a significant decrease in arachidonate/linoleate ratios of hepatic PE, PS-PI and PC, while total phospholipid content remained constant. In the high-fat, ethanol-treated group, hepatic cholesterol content was increased 2-fold. These results suggest that the level of dietary fat plays an important role in determining the effects of chronic ethanol consumption on hepatic cholesterol content and phospholipid acyl composition.     

Purification of surfactant from lung washings and washings contaminated with blood constituents

A rapid and simple method capable of purifying surfactant from rabbit alveolar washings and from washings contaminated with serum has been developed. The sample, containing 16% NaBr, is placed beneath a two-layer discontinuous gradient of NaBr. After centrifugation, the surfactant is found near the top of the gradient tube at a density of 1.085 at 4 C while the contaminating material remains near the bottom. The lipid composition of surfactant from lung washings of normal animals isolated by this method compares quite favorably with surfactant isolated by much more elaborate and time consuming methods. Surfactant purified from mixtures of³H-palmitate labeled rabbit serum and lung washings (1.6 mg serum phospholipid: 1 mg washing phospholipid) contained less than 3% of the phospholipid radioactivity. The phospholipid composition of this band was quite similar to that of surfactant from normal lung washings, but the protein content was much higher. A second density gradient centrifugation removed 90% of this protein resulting in a surfactant fraction with a phospholipid to protein ratio similar to that of surfactant from normal lung washings. These findings demonstrate that this purification method is capable of removing a large proportion of both serum phospholipids and proteins from lung washings contaminated with serum, making this method uniquely suitable for evaluation of surfactant in pathologic conditions of the lung.     

Phospholipid exchange between subcellular organelles of rabbit lung

A soluble protein fraction (PLEP) prepared from rabbit lung can catalyze the exchange of phospholipids between subcellular organelles of the lung and between these subcellular organelles and synthetic liposomes. Phospholipid exchange between microsomes and synthetic liposomes and between mitochondria and synthetic liposomes was stimulated 8-fold and 2.5-fold, respectively, in the presence of the protein fraction. Lung exchange protein could also catalyze phospholipid exchange between subcellular organelles of the liver and synthetic liposomes. Phospholipid transfer between microsomes and lamellar bodies of the lung was stimulated 2-fold by the exchange protein. Both radiolabeled phosphatidylcholine (PC) and phosphatidylinositol (PI) were transferred from³²P-labeled microsomes to lamellar bodies, but the exchange protein exhibited no transfer activity for phosphatidylglycerol (PG) and that for phosphatidylethanolamine (PE) was insignificant compared to the transfer activity for phosphatidylcholine and phosphatidylinositol. While the physiological role of the phospholipid exchange proteins in the lung is unknown, it is possible that they participate in the distribution of the newly synthesized phospholipids from the site of synthesis to lamellar bodies and other membrane compartments of cells.     

Alveolar lavage and lavaged lung tissue phosphatidylcholine composition during fetal rabbit development

Pulmonary surfactant, the major surface-active component of which in the adult is dipalmitoylglycerophosphocholine, can be obtained by lavaging lungs with physiological saline. We have previously shown that there is an increase in the amount of phosphatidylcholine in fetal rabbit lung lavage during the latter part of gestation. We have now measured the amount of disaturated phosphatidylcholine as well as the fatty acid composition of phosphatidylcholine in lung lavage from fetal rabbits during the period 27 days’ gestation to full term (31 days). There was no developmental change in the amount of disaturated phosphatidylcholine during the period examined. About 50% of the total phosphatidylcholine was disaturated which is approximately the same as in adult rabbit lung lavage. The fatty acid composition, however, did change. There was an increase in the amount of 16∶0 from about 20% of the total fatty acids in phosphatidylcholine at 27–28 days to about 60% at full term, after birth, and in the adults. There was a corresponding decrease in the amounts of 14∶0, 18∶0, and longer chain fatty acids, most of which were saturated. In the lavaged lung tissue, there was a 2.6-fold increase in the percentage of phosphatidylcholine which was disaturated during the period 27–31 days’ gestation. It had not decrease to the adult value 24 hr after birth.     

Phase behavior and hydrated solid structure in lysophospholipid/long-chain alcohol/water system and effect of cholesterol addition

Phase behavior in lysophospholipid/long-chain alcohol/water system at 80°C was investigated using hexanol and oleyl alcohol as the long-chain alcohol. Similarly to hydrophilic surfactant, a micellar phase in a lysophospholipid/water system transitioned to a lamellar liquid-crystalline phase by the addition of long-chain alcohol. In the oleyl alcohol system the lamellar liquid-crystalline phase was observed in wider region compared to the hexanol system. The effect of cholesterol addition on the phase behavior was also studied. The region of liquid-crystalline phase and (reverse micellar + liquid-crystalline + water) phase shifted towards higher lysophospholipid concentrations. The structure of hydrated solid as well as the transition between lamellar liquid-crystalline phase and hydrated solid was analyzed by X-ray scattering measurement and differential scanning calorimetry measurement. It was revealed that the hydrated solid was α-type crystals with lamellar structure. The hydrated solid (gel)-liquid crystal transition temperature gradually decreased with increasing oleyl alcohol concentration and the decrement was enhanced by the addition of cholesterol.