L.M.G. Van Golde

The Pulmonary Surfactant System: Biochemical and Clinical Aspects

Abstract. This article starts with a brief account of the history of research on pulmonary surfactant. We will then discuss the morphological aspects and composition of the pulmonary surfactant system. We describe the hydrophilic surfactant proteins A and D and the hydrophobic surfactant proteins B and C, with focus on the crucial roles of these proteins in the dynamics, metabolism, and functions of pulmonary surfactant. Next we discuss the major disorders of the surfactant system. The final part of the review will be focused on the potentials and complications of surfactant therapy in the treatment of some of these disorders. It is our belief that increased knowledge of the surfactant system and its functions will lead to a more optimal composition of the exogenous surfactants and, perhaps, widen their applicability to treatment of surfactant disorders other than neonatal respiratory distress syndrome.

The effect of dietary fat on the molecular species of lecithin from rat liver

Abstract 1. 1. Lecithins from the liver of rats maintained on diets devoid of essential fatty acids or supplemented with coconut oil or corn oil revealed significant differences in fatty acid composition, whilst monomolecular films of these lecithin samples exhibited only limited differences in force-area characteristics. 2. 2. The individual molecular species in the three lecithin samples were determined by means of the following techniques: (a) fractionation of the lecithin on silica impregnated with silver nitrate, followed by determination of the positional distribution of the fatty acids with snake-venom phospholipase A (EC 3.1.1.4); (b) hydrolysis of the lecithin with phospholipase C (EC 3.1.4.3) from Bacillus cereus and fractionation of the resultant diglycerides on silica impregnated with silver nitrate. Subsequently, the positions of the fatty acid constituents in the diglyceride fractions were determined by hydrolysis with pancreatic lipase (EC 3.1.1.3). The second approach gave the most detailed information and made it possible to recognize between 14 and 23 species. Quantitative determination of the major species accounted for 78.5 to 90.5% of the molecular composition. 3. 3. Differences in diet induced significant variations in the proportions of the various lecithin species and also brought about qualitative differences. These shifts in the molecular composition of the lecithin samples may contribute to preserving the liquid-crystalline nature of these lipids in membrane structures.

Some studies on the metabolism of phospholipids in Golgi complex from bovine and rat liver in comparison to other subcellular fractions

Abstract 1. 1. Golgi complex, rough and smooth microsomes, plasma membranes, mitochondria, and nuclei from bovine liver were isolated and the purity assessed using specific marker enzymes. 2. 2. Cholinephosphotransferase and acyl-CoA:1,2-diacyl-sn-glycerol acyltransferases were found to be absent in the Golgi complex, mitochondria, plasma membrane, and nuclei. The de novo synthesis of lecithin and triacylglycerols is apparently localized exclusively in the smooth and rough endoplasmic reticulum. 3. 3. Acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase was found to be localized mainly in rough and smooth microsomes from bovine and rat liver; though also significant activity was found in the Golgi complex of bovine liver. On the other hand, no significant acylation of 1-acyl-sn-glycero-3-phosphorylcholine could be shown in the Golgi system from rat liver. 4. 4. Phosphatidylglycerol synthesis was found to take place predominantly in rat liver mitochondria. Significant synthesis of this phospholipid was, however, also found in rough and smooth microsomes and in Golgi of rat liver. 5. 5. The Golgi complex of rat liver was shown to contain both phospholipase A1 and A2 activities acting on exogenous phosphatidylethanolamine, the activity of the former being somewhat larger.

POSITIONAL SPECIFICITY OF SATURATED AND UNSATURATED FATTY ACIDS IN PHOSPHATIDIC ACID FROM RAT LIVER

Abstract 1. 1. The relative incorporation of a number of radioactive fatty acids into the different glycerolipids of rat liver microsomes has been investigated. 2. 2. Studies on the distribution of the radioactivity incorporated into phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid showed that in all three lipids, the majority of the saturated fatty acids were at the 1-position while the polyunsaturated fatty acids were largely confined to the 2-position. 3. 3. The phosphatidic acid fraction of rat liver was isolated in a pure form, and its fatty acid distribution agreed with the results of the incorporation studies. 4. 4. The incorporation of radioactive fatty acids into different molecular species of phosphatidic acid, separated as dimethyl phosphatidates was investigated.

Biosynthesis of lipids in golgi complex and other subcellular fractions from rat liver

Abstract 1. 1. Golgi complex, rough and smooth microsomes, plasma membranes, mitochondria and nuclei from rat liver were isolated and their purity assessed using specific marker enzymes. 2. 2. The various subcellular fractions were assayed for the following processes: biosynthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine, the conversion of phosphatidylserine into phosphatidylethanolamine, the formation of lecithin via N-methylation, and the activation of palmitic and octanoic acids. 3. 3. None of these processes were found to be present in Golgi complex. 4. 4. The endoplasmic reticulum appears to be the principal site in the cell for the synthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine and the formation of lecithin. Interestingly, the biosynthesis of phosphatidylserine appears to be four times more active in rough than in smooth microsomes, which might suggest a ribosomal localization of this process. 5. 5. Except for CDPdiglyceride synthesis, mitochondria do not contain any of the synthesizing activities described in 4. Mitochondria are, however, the only site in the cell where phosphatidylserine is decarboxylated. This activity appears to be localized in the inner membrane. 6. 6. The activation of palmitate is localized predominantly in endoplasmic reticulum and mitochondria, though some activity was detected in plasma membranes as well. All other cell organelles, including Golgi and probably nuclei, did not contain significant palmitoyl-CoA synthetase activity. The subcellular distribution of octanoyl-CoA synthetase resembled that of palmitoyl-CoA synthetase except that the former enzyme is more active in mitochondria than in microsomes.

Molecular species of lecithins from various animal tissues.

Abstract 1. 1. The molecular species composition of lecithins from lung, brain, liver and kidney derived from rat, rabbit, pig and cow was determined. 2. 2. In all animals considerable differences were found in the molecular composition of lecithins from the various tissues. The composition of lecithins from lung and brain showed many similarities irrespective of animal species. This may indicate a certain degree of tissue specificity. On the other hand, pronounced differences were observed in the lecithin composition of liver and kidney from the different animals. 3. 3. (Dipalmitoyl)-lecithin was the main lecithin present in lung, but was only a trace compound in the liver and kidney of all animals examined. No significant amounts of (distearoyl)- or (1-palmitoyl-2-stearoyl)-lecithin could be detected in any tissues investigated. 4. 4. Lecithin species containing oleic acid were found to predominate in brain. In general, the content of (1-palmitoyl-2-oleoyl)-lecithin was higher than (1-stearoyl-2-oleoyl)-lecithin. Species with linoleic acid were not significantly present in brain. 5. 5. Lecithins containing one saturated and one polyunsaturated fatty acid constituent are abundant in the liver and kidney of all animals species examined. Molecules with two identical or two different polyunsaturated acyl constituents were not encountered in significant amounts in the tissues investigated.

Some studies on the biosynthesis of the molecular species of phosphatidylcholine from rat lung and phosphatidylcholine and phosphatidylethanolamine from rat liver.

Abstract 1. 1. At different time intervals after injection of [ I (3)- 3 H]glycerol, the incorporation of glycerol into the various molecular species of phosphatidylcholine and phosphatidylethanolamine from rat liver, and phosphatidylcholine from rat lung was determined. 2. 2. The results indicate that, in liver, a de novo synthesis is primarily operating in the biosynthesis of linoleic acid-containing molecules of lecithin and of the hexaenoic molecular species of phosphatidylethanolamine. An acylation of monoacyl derivatives of these phospholipids is suggested to play an important role particularly in the formation of arachidonic acid containing molecular species of these phospholipids. 3. 3. In lung, the de novo synthesis was found to contribute also primarily to the linoleic acid-containing lecithins, though it also represents an important pathway for the synthesis of tetraenoic, monoenoic and, perhaps to a lesser extent, disaturated lecithins. A deacylation-reacylation mechanism may contribute significantly to the formation of dipalmitoyl lecithin, a major constituent of lung pulmonary surfactant. 4. 4. Acylation of I -palmitoyl- sn -glycero-3-phosphorylcholine with various labeled fatty acids or acyl-CoA esters was studied in the presence of microsomes from rat lung and liver. In the presence of microsomes from lung a significant uptake of palmitic acid was observed into the 2-position of lecithin, this in strong contrast to the findings with liver microsomes where only a very limited uptake of palmitic acid was observed. The results endorse the findings from the in vivo studies that the acylation of monoacyl- sn -glycero-3-phosphorylcholine may play an important additional role in maintaining the high level of dipalmitoyl lecithin in lung. 5. 5. Comparison of the composition of phosphatidylethanolamine and lecithin from lung suggested, in support of previous observations by other investigators, that the methylation of phosphatidylethanolamine does not represent an important pathway for the formation of dipalmitoyl lecithin.

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.

The intramitochondrial distribution of some enzymes involved in the biosynthesis of rat-liver phospholipids

Abstract 1. 1. The conversions of 1- and 2-acyl-sn-glycero-3-phosphorylcholine into 3-sw-phosphatidylcholine, of i-acyl-sw-glycero-3-phosphate into i,2-diacyl-sn-glycero-3-phosphate, and of 1,2 diacyl-sw-glycerols into 3-sw-phosphatidylcholine and triacyl-glycerols were stud microsomes, mitochondria and submitochondrial fractions from rat liver and compared with the distribution of glucose-6-phosphatase, rotenone-insensitive NADH-cytochrome c reductase, cytochrome oxidase and succinate dehydrogenase. 2. 2. The information obtained strongly indicates that mitochondria do contain the acyltransferases involved in the conversion of both isomeric mono-acyl-sn-glycero-3-phosphorylcholines into 3-sw-phosphatidylcholine and of acyl-sn-glycero-3-phosphate into 1,2-diacyl-sn-glycero-3-phosphate. These processes were found to occur predominantly in the outer membrane fraction. 3. 3. Mitochondria and mitochondrial subfractions did not catalyze significantly the conversion of 1,2-diacyl-sn-glycerols with [14C]CDP-choline into 3-sn-phosphatidylcholine or that of 1,2-diacyl-sn-glycerols with [14C]fatty acids into triacylglycerols, this in strong contrast with microsomes. The specific activities of these enzymes are actually much lower than that of glucose-6-phosphatase in the submitochondrial fractions. 4. 4. Simultaneous incubation of sn-[2-3H]glycero-3-phosphate and [14C]fatty acids with submitochondrial fractions demonstrated that sn-glycero-3-phosphate was converted, in the presence of outer membranes, into 1,2-diacyl-sn-glycero-3-phosphate. However, the small3H/14C ratios observed in 3-sn-phosphatidylcholine and phosphatidylethanolamine allow the conclusion that, at least under these conditions, the uptake of fatty acids into these phospholipids proceeds mainly via acylation of their mono-acyl derivatives.

Preferential incorporation of fatty acids at the inside of human erythrocyte membranes

1. 1.Phospholipase A2 isolated from Naja naja venom was used as a tool to discriminate between the outer and inner lipid monolayer of the membranes of human erythrocytes. 2. 2.Incubation of human erythorcytes with radioactive fatty acids resulted in the formation of labelled lecithin in the membrane. The label was found predominantly in that pool of lecithin which is localized in the inner monolayer of the membrane. 3. 3.Lecithins isolated from the total erythrocyte membrane and from the inner monolayer of the membrane possess identical fatty acid patterns and identical positional distributions of their fatty acyl constituents.