Blocem J.- (1975) 151, 707-714 Printed in Great Britain
707
Characterization of Pulmonary Surfactant from Ox, Rabbit, Rat and Sheep By JOHN L. HARWOOD, RASHMI DESAI, PAUL HEXT, TERESA TETLEY and ROY RICHARDS Department of Biochemistry, University College, P.O. Box 78, Cardiff CFI 1XL, U.K.
(Received 17 July 1975) 1. Pulmonary surfactants from ox,'rabbit, rat and sheep were isolated and analysed. 2. All preparations had a high anenoic phosphatidylcholine content and would produce stable surface tensions of 0.01 N m1 or less. 3. Protein content was 8-18% of the dry weights. A number of proteins were observed; their overall compositions were high in hydrophobic amino acid residues. 4. Lipid content varied from 79% (ox) to 90% (rabbit) with phosphatidylcholine representing from 58% (sheep) to 83 % (rabbit) of the total lipid. The surfactant preparations were rather similar in lipid composition except that sheep surfactant contained about 10% lysophosphatidylcholine. 5. Hexadecanoic acid was the principal fatty acid. It was particularly high in phosphatidylcholine. 6. Phosphatidylglycerol was a minor constituent of all surfactants but phosphatidyldinethylethanolamine was not detected. Pulmonary surfactant is a complex mixture of substances which is responsible for the unusually high surface activity present in mammalian lungs. Only recently has the vital role of this lipoprotein in normal and abnormal lung function been fully recognized. Notably, its deficiency is the cause of acute respiratory distress syndrome (Gluck et al., 1972) and it increases in amount remarkably during exposure of animals to quartz dust (Heppleston et al., 1974) and in human alveolar proteinosis (Ramirez-R & Harlan, 1968). The properties and metabolism of the surfactant have been reviewed (Goerke, 1974; King, 1974; Tierney, 1974). Although several components of pulmonary surfactant have the ability to develop extremely low surface tensions (less than 0.01N.m-1) it is usually considered that dipalmitylphosphatidylcholine is the most important (King & Clements, 1972a,b). This particular phospholipid is present in an unusually high concentration in canine pulmonary surfactant (King & Clements, 1972b). Most workers (see King 1974; Tierney, 1974) consider that protein is an important, if minor, component of the surfactant but Scarpelli et al. (1970) and Steim et al. (1969) claim that no protein is present. King et al. (1973) have suggested that one protein can be considered as being unique to surfactant and this together with other proteins, may play a role in the secretion of surfactant into the alveolar spaces (Goerke, 1974). Finally, the other lipid constituents and the unsaturated molecular species of phosphatidylcholine may help to speed the spreading of the surfactant layer within the alveolus (King, 1974). Vol. 151
Few of the analyses of surfactant have been very detailed but have largely concentrated on particular aspects such as phosphatidylcholine or protein content (e.g. Abrams, 1966; Pruitt et al., 1971). As part of a study on the role of surfactant in lung function we have now carried out a detailed analysis of surfactants obtained from different mammalian species. Although each surfactant contains high concentrations of dipalmitoyl phosphatidylcholine and is capable of generating low, stable surface tensions, there are significant differences in chemical composition between types. These variations are manifested in differences in physical properties. MAterials and Methods Fatty acid, triacylglycerol, cholesterol, sphingomyelin, phosphatidylcholine and phosphatidylethanolamine standards were purchased from Sigma (London) Chemical Co. Ltd., Kingston-uponThames, Surrey KT2 7BH, U.K. Lipid standards were found to be homogeneous by t.l.c. and the fatty acids were found to be homogeneous by g.l.c. Phosphatidylinositol, purchased from Koch-Light Laboratories Ltd., Colnbrook, Bucks., U.K., was purified by t.l.c. Lysophosphatidylcholine, was prepared from egg phosphatidylcholine by the action of Rhizopus arrhizus phospholipase A [Boehringer (London) Corp. Ltd., Lewes, Sussex BN7 1LG, U.K.] by using the incubation conditions described by Tulloch et al. (1973). Phosphatidylglycerol was isolated from broad-bean leaves as described by Harwood & James (1975). Other reagents were of the
708
J. L. HARWOOD, R. DESAI, P. HEXT, T. TETLEY AND R. RICHARDS
highest available grades and were purchased from BDH Chemicals Ltd. (Poole, Dorset, U.K.), from Boehringer, and from Sigma. Ox and sheep lungs were obtained direct from the slaughterhouse with the co-operation of Mr. W. G. Lewis. Post-mortem delay was kept to a minimum and lung lavage was carried out within 1 h of the death of the animal. Dutch rabbits were used from the departmental breeding stock with no post-mortem delay. Specific pathogen-free rats were kindly supplied by the MRC Pneumoconiosis Research Unit, Llandough Hospital, Cardiff, U.K. Isolation of surfactant Pulmonary surfactant was obtained by a modification of the method of Abrams (1966). After lung lavage with iso-osmotic NaCl (Brain, 1970), the free cell population was removed by centrifugation (300g for 20min). The supernatant was centrifuged at 10OOg for 60min and the pellet was resuspended in 21 % (w/v) NaCl. Further centrifugation at 15OOg for 25min resulted in a three-phase separation into an insoluble floating fraction (surfactant pellicle), a soluble fraction (soluble surfactant) and a small precipitate at the bottom of the tube. The surfactant pellicle fraction was dialysed against water for 48h (Desai et al., 1975). All the above procedures were carried out at 4°C. The pellicle surfactant was freezedried and stored under N2 at -15°C. Protein separation and analysis Protein components of the isolated surfactants were separated by polyacrylamide-gel electrophoresis by using 5.8% (w/v) gels to which the sample was added in membrane solvent (Fairbanks et al., 1971). The pH of the running buffer was 7.4. Alternatively, the surfactant sample was delipidated by the method of Scanu et al. (1969) before electrophoresis. In both cases the gels were stained after electrophoresis with 0.05 % (w/v) Coomassie Blue. Protein was estimated by the Lowry et al., (1951) method as modified by Oyama & Eagle (1956). Bovine serum albumin was used as standard. The amino acid composition of the surfactants was measured on 6M-HCI hydrolysates in a Technicon amino acid autoanalyser (Technicon Instruments Ltd., New York, N.Y., U.S.A.). Tryptophan was not measured.
Lipid extraction, separation and estimation Lipids were extracted from the surfactant samples by the method of Garbus et al. (1963). They were separated by t.l.c. on silica gel G (E. Merck, Darmstadt, Germany) plates in chloroform-methanolacetic acid-water (170:30:20:7, by vol.). This solvent separated all the principal phospholipid components. For the separation of lysophosphatidylcholine from phosphatidylinositol a solvent of
chloroform-methanol-water (14:6:1, by vol.) was used and, for neutral lipids, one of light petroleum (b.p. 40-60'C)-diethyl ether-acetic acid (90:10:1, by vol.) was used. Lipid bands were revealed with I2 vapour or aq. 0.01 % (w/v) Rhodamine 6G (when fatty acids were to be analysed). Phospholipids were provisionally identified by co-chromatography with standards and by differential colour reactions with phosphate, ninhydrin and Dragendorff sprays. They were also eluted from the t.l.c. plates with chloroformmethanol-acetic acid (200:100:1, by vol.) and were identified after paper chromatography of their deacylated derivatives (Dawson, 1960) and acid hydrolysates (Harwood & Stumpf, 1970). Fatty acid components were analysed by g.l.c. after boron trifluoride-methanol transesterification. An internal standard of pentadecanoic acid was added for quantitation. Separations were achieved in 15% (w/w) diethylene glycol succinate on Chromosorb W AW (80-100 mesh; Supleco Inc., Bellefont, Pa. 16823, U.S.A.) or in 15 % (w/w) polyethylene glycol adipate on Diatomite CS (60-72 mesh; Pye Unicam Ltd., Cambridge, U.K.) by using either isothermal or temperature programming in the range 150-182°C on a Perkin-Elmer Fl1, a Perkin-Elmer F33 or a Pye 104 gas chromatogram. The positions of the double bonds of unsaturated fatty acids were not determined. Phosphorus was analysed by the method of Bartlett (1959), total esters by the method of Stern & Shapiro (1953) and cholesterol by the method of Rodnight (1957). Surface-tension measurements The surface tensions of surfactant layers were measured in iso-osmotic NaCl by using a LangmuirWilhelmy balance. Samples (400ag/ml of ethanol) were added 8,ug at a time until no change in surface tension was observed. A compression-decompression cycle was then performed. Measurements were carried out at 20 and 37°C and each sample was measured at least in triplicate. Results and Discussion
King (1974) discussed at some length the difficulty of characterizing the surface-active material isolated from lungs, and recommended a number of properties that should be present in any material called 'pulmonary surfactant'. These include the ability to lower surface tension of a Ringer solution at 37°C to 0.01 N m- or less, and the enrichment of dipalmitoyl phosphatidylcholine. Goerke (1974) also emphasized similar characteristics. King (1974) also suggested that the best preparations of pulmonary surfactant are obtained after gentle lavage and a series of centrifugations of the resulting fluid. Mindful of the 1975
709
PULMONARY SURFACTANT FROM DIFFERENT MAMMALS Table 1. Lipid andprotein contents of surfactants isolatedfrom different mammals For details see the text. Means ± S.D. are given with the number of experiments in parentheses. Lipid or protein content Animal
...
Rat
Rabbit
Starting material
...
Wash
Wash 8± 1 (2)
Sheep
Ox
HomoWash
genate
Wash
18±1 (2) 33 ±2 (2) 14±1 (3) 10±1 (3) 88±1 (3) 90±3 (2) 79±7 (2) 63±5 (2) 86± 3 (3) 1.21 (1) 0.78±0.03 (3) 0.081 ±0.08 (2)
Protein (%, w/w) Lipid (%/, w/w) Yield (mg/g fresh wt. of lung)
Table 2. Amino acid composition of different surfactants Results are expressed as ,mol/mg dry wt. of surfactant. tr, Trace (
707
Characterization of Pulmonary Surfactant from Ox, Rabbit, Rat and Sheep By JOHN L. HARWOOD, RASHMI DESAI, PAUL HEXT, TERESA TETLEY and ROY RICHARDS Department of Biochemistry, University College, P.O. Box 78, Cardiff CFI 1XL, U.K.
(Received 17 July 1975) 1. Pulmonary surfactants from ox,'rabbit, rat and sheep were isolated and analysed. 2. All preparations had a high anenoic phosphatidylcholine content and would produce stable surface tensions of 0.01 N m1 or less. 3. Protein content was 8-18% of the dry weights. A number of proteins were observed; their overall compositions were high in hydrophobic amino acid residues. 4. Lipid content varied from 79% (ox) to 90% (rabbit) with phosphatidylcholine representing from 58% (sheep) to 83 % (rabbit) of the total lipid. The surfactant preparations were rather similar in lipid composition except that sheep surfactant contained about 10% lysophosphatidylcholine. 5. Hexadecanoic acid was the principal fatty acid. It was particularly high in phosphatidylcholine. 6. Phosphatidylglycerol was a minor constituent of all surfactants but phosphatidyldinethylethanolamine was not detected. Pulmonary surfactant is a complex mixture of substances which is responsible for the unusually high surface activity present in mammalian lungs. Only recently has the vital role of this lipoprotein in normal and abnormal lung function been fully recognized. Notably, its deficiency is the cause of acute respiratory distress syndrome (Gluck et al., 1972) and it increases in amount remarkably during exposure of animals to quartz dust (Heppleston et al., 1974) and in human alveolar proteinosis (Ramirez-R & Harlan, 1968). The properties and metabolism of the surfactant have been reviewed (Goerke, 1974; King, 1974; Tierney, 1974). Although several components of pulmonary surfactant have the ability to develop extremely low surface tensions (less than 0.01N.m-1) it is usually considered that dipalmitylphosphatidylcholine is the most important (King & Clements, 1972a,b). This particular phospholipid is present in an unusually high concentration in canine pulmonary surfactant (King & Clements, 1972b). Most workers (see King 1974; Tierney, 1974) consider that protein is an important, if minor, component of the surfactant but Scarpelli et al. (1970) and Steim et al. (1969) claim that no protein is present. King et al. (1973) have suggested that one protein can be considered as being unique to surfactant and this together with other proteins, may play a role in the secretion of surfactant into the alveolar spaces (Goerke, 1974). Finally, the other lipid constituents and the unsaturated molecular species of phosphatidylcholine may help to speed the spreading of the surfactant layer within the alveolus (King, 1974). Vol. 151
Few of the analyses of surfactant have been very detailed but have largely concentrated on particular aspects such as phosphatidylcholine or protein content (e.g. Abrams, 1966; Pruitt et al., 1971). As part of a study on the role of surfactant in lung function we have now carried out a detailed analysis of surfactants obtained from different mammalian species. Although each surfactant contains high concentrations of dipalmitoyl phosphatidylcholine and is capable of generating low, stable surface tensions, there are significant differences in chemical composition between types. These variations are manifested in differences in physical properties. MAterials and Methods Fatty acid, triacylglycerol, cholesterol, sphingomyelin, phosphatidylcholine and phosphatidylethanolamine standards were purchased from Sigma (London) Chemical Co. Ltd., Kingston-uponThames, Surrey KT2 7BH, U.K. Lipid standards were found to be homogeneous by t.l.c. and the fatty acids were found to be homogeneous by g.l.c. Phosphatidylinositol, purchased from Koch-Light Laboratories Ltd., Colnbrook, Bucks., U.K., was purified by t.l.c. Lysophosphatidylcholine, was prepared from egg phosphatidylcholine by the action of Rhizopus arrhizus phospholipase A [Boehringer (London) Corp. Ltd., Lewes, Sussex BN7 1LG, U.K.] by using the incubation conditions described by Tulloch et al. (1973). Phosphatidylglycerol was isolated from broad-bean leaves as described by Harwood & James (1975). Other reagents were of the
708
J. L. HARWOOD, R. DESAI, P. HEXT, T. TETLEY AND R. RICHARDS
highest available grades and were purchased from BDH Chemicals Ltd. (Poole, Dorset, U.K.), from Boehringer, and from Sigma. Ox and sheep lungs were obtained direct from the slaughterhouse with the co-operation of Mr. W. G. Lewis. Post-mortem delay was kept to a minimum and lung lavage was carried out within 1 h of the death of the animal. Dutch rabbits were used from the departmental breeding stock with no post-mortem delay. Specific pathogen-free rats were kindly supplied by the MRC Pneumoconiosis Research Unit, Llandough Hospital, Cardiff, U.K. Isolation of surfactant Pulmonary surfactant was obtained by a modification of the method of Abrams (1966). After lung lavage with iso-osmotic NaCl (Brain, 1970), the free cell population was removed by centrifugation (300g for 20min). The supernatant was centrifuged at 10OOg for 60min and the pellet was resuspended in 21 % (w/v) NaCl. Further centrifugation at 15OOg for 25min resulted in a three-phase separation into an insoluble floating fraction (surfactant pellicle), a soluble fraction (soluble surfactant) and a small precipitate at the bottom of the tube. The surfactant pellicle fraction was dialysed against water for 48h (Desai et al., 1975). All the above procedures were carried out at 4°C. The pellicle surfactant was freezedried and stored under N2 at -15°C. Protein separation and analysis Protein components of the isolated surfactants were separated by polyacrylamide-gel electrophoresis by using 5.8% (w/v) gels to which the sample was added in membrane solvent (Fairbanks et al., 1971). The pH of the running buffer was 7.4. Alternatively, the surfactant sample was delipidated by the method of Scanu et al. (1969) before electrophoresis. In both cases the gels were stained after electrophoresis with 0.05 % (w/v) Coomassie Blue. Protein was estimated by the Lowry et al., (1951) method as modified by Oyama & Eagle (1956). Bovine serum albumin was used as standard. The amino acid composition of the surfactants was measured on 6M-HCI hydrolysates in a Technicon amino acid autoanalyser (Technicon Instruments Ltd., New York, N.Y., U.S.A.). Tryptophan was not measured.
Lipid extraction, separation and estimation Lipids were extracted from the surfactant samples by the method of Garbus et al. (1963). They were separated by t.l.c. on silica gel G (E. Merck, Darmstadt, Germany) plates in chloroform-methanolacetic acid-water (170:30:20:7, by vol.). This solvent separated all the principal phospholipid components. For the separation of lysophosphatidylcholine from phosphatidylinositol a solvent of
chloroform-methanol-water (14:6:1, by vol.) was used and, for neutral lipids, one of light petroleum (b.p. 40-60'C)-diethyl ether-acetic acid (90:10:1, by vol.) was used. Lipid bands were revealed with I2 vapour or aq. 0.01 % (w/v) Rhodamine 6G (when fatty acids were to be analysed). Phospholipids were provisionally identified by co-chromatography with standards and by differential colour reactions with phosphate, ninhydrin and Dragendorff sprays. They were also eluted from the t.l.c. plates with chloroformmethanol-acetic acid (200:100:1, by vol.) and were identified after paper chromatography of their deacylated derivatives (Dawson, 1960) and acid hydrolysates (Harwood & Stumpf, 1970). Fatty acid components were analysed by g.l.c. after boron trifluoride-methanol transesterification. An internal standard of pentadecanoic acid was added for quantitation. Separations were achieved in 15% (w/w) diethylene glycol succinate on Chromosorb W AW (80-100 mesh; Supleco Inc., Bellefont, Pa. 16823, U.S.A.) or in 15 % (w/w) polyethylene glycol adipate on Diatomite CS (60-72 mesh; Pye Unicam Ltd., Cambridge, U.K.) by using either isothermal or temperature programming in the range 150-182°C on a Perkin-Elmer Fl1, a Perkin-Elmer F33 or a Pye 104 gas chromatogram. The positions of the double bonds of unsaturated fatty acids were not determined. Phosphorus was analysed by the method of Bartlett (1959), total esters by the method of Stern & Shapiro (1953) and cholesterol by the method of Rodnight (1957). Surface-tension measurements The surface tensions of surfactant layers were measured in iso-osmotic NaCl by using a LangmuirWilhelmy balance. Samples (400ag/ml of ethanol) were added 8,ug at a time until no change in surface tension was observed. A compression-decompression cycle was then performed. Measurements were carried out at 20 and 37°C and each sample was measured at least in triplicate. Results and Discussion
King (1974) discussed at some length the difficulty of characterizing the surface-active material isolated from lungs, and recommended a number of properties that should be present in any material called 'pulmonary surfactant'. These include the ability to lower surface tension of a Ringer solution at 37°C to 0.01 N m- or less, and the enrichment of dipalmitoyl phosphatidylcholine. Goerke (1974) also emphasized similar characteristics. King (1974) also suggested that the best preparations of pulmonary surfactant are obtained after gentle lavage and a series of centrifugations of the resulting fluid. Mindful of the 1975
709
PULMONARY SURFACTANT FROM DIFFERENT MAMMALS Table 1. Lipid andprotein contents of surfactants isolatedfrom different mammals For details see the text. Means ± S.D. are given with the number of experiments in parentheses. Lipid or protein content Animal
...
Rat
Rabbit
Starting material
...
Wash
Wash 8± 1 (2)
Sheep
Ox
HomoWash
genate
Wash
18±1 (2) 33 ±2 (2) 14±1 (3) 10±1 (3) 88±1 (3) 90±3 (2) 79±7 (2) 63±5 (2) 86± 3 (3) 1.21 (1) 0.78±0.03 (3) 0.081 ±0.08 (2)
Protein (%, w/w) Lipid (%/, w/w) Yield (mg/g fresh wt. of lung)
Table 2. Amino acid composition of different surfactants Results are expressed as ,mol/mg dry wt. of surfactant. tr, Trace (
