Lung (1988)166:293-301

New York Inc. 1988

Changes in Phospholipids in Bronchoalveolar Lavage Fluid of Patients with Interstitial Lung Diseases Yasuhito Honda, ~-* Kazunori Tsunematsu, t Akira Suzuki, ~ and Toyoaki Akino 2 ~Third Department of Internal Medicine and '-Department of Biochemistry, Sapporo Medical College, Sapporo, Japan

Abstract. We analyzed phospholipids of human bronchoalveolar lavage (BAL) fluids from patients with interstitial lung diseases; idiopathic pulmonary fibrosis (IPF), sarcoidosis, and eosinophilic granuloma (EG) and compared them to those of normal subjects. The content of phospholipid/ml of BAL fluid was significantly decreased in IPF. There was a significant decrease in phosphatidylglycerol (PG) and an increase in phosphatidylinositol (PI) in IPF but not in sarcoidosis and EG. Thus, the PG to PI ratio was significantly decreased in IPF. The dipalmitoyl species of phosphatidylcholine (PC) was found to be significantly decreased in IPF and sarcoidosis by molecular species analysis using high performance liquid chromatography. In contrast, the unsaturated species were increased in these diseases. The decrease in dipalmitoyl PC appeared to be a common feature in interstitial lung diseases. The changes in phospholipids in BAL fluids, especially decreases in DPPC and PG to PI ratio in IPF, appear to indicate that damage of alveolar Type II cells and/or of metabolic disturbance in pulmonary surfactant occurs in IPF. Key words: Interstitial lung disease--Bronchoalveolar lavage fluid--Pulmonary surfactant--Phospholipid--Dipalmitoyl phosphatidylcholine. Introduction Bronchoalveolar lavage (BAL), a method of sampling lower airway secretions, has been routinely used in the cytological and biochemical evaluation of pulmonary diseases. Phospholipids in BAL fluid have been analyzed to elucidate * To whom offprint requests should be addressed at Third Department of Internal Medicine, Sapporo Medical College, Chuo-ku, S 1. W. 17, Sapporo 060, Japan

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changes in pulmonary surfactant in various pulmonary diseases, and specific changes in the phospholipid profiles have been reported in alveolar proteinosis [2, 20, 25], adult respiratory distress syndrome [7, 14], bacterial pneumonia [7], and sarcoidosis [8]. Pulmonary surfactant, which mainly consists of phospholipids such as dipalmitoyl phosphatidylcholine (DPPC) and phosphatidylglycerol (PG) [1] is synthesized in alveolar Type II cells and secreted into alveolar spaces, where it stabilizes the pulmonary alveoli against collapse [11]. Recent observations suggest that phospholipid metabolism in alveolar Type II ceils seems to be affected by chemical mediators derived from other lung cells [27]. Therefore, phospholipid analysis of BAL fluid is important in evaluating the states of alveolar Type II cells and metabolic changes in pulmonary surfactant in lung diseases. The relationship between pulmonary surfactant metabolism and interstitial lung diseases remains obscure and there have been only a few reports on BAL-phospholipids of interstitial lung diseases [8, 20]. In the present study, we analyzed phospholipids in BAL fluid from patients with interstitial lung diseases in detail using modern methods established recently. Materials and Methods

Materials A total of 13 patients (6 with IPF, 4 with sarcoidosis, and 3 with EG) and 8 healthy volunteers were included in this study. Ten were smokers and 11 were nonsmokers. Phospholipids in BAL fluids from smokers were not significantly different from those from nonsmokers. All of the patients were diagnosed according to clinical symptoms, roentgen01ogical findings and physiological studies, including the analysis of lung biopsy specimens. None received prednisolone [23] and ambroxol [22], which affect the metabolism of phospholipids in the lung.

Bronchoalveolar Lavage All bronchoscopies were performed with a fiberoptic bronchoscope (Type 4B-2, Olympus). All cases were premedicated with intramuscular atropine (0.5 mg) and either hydroxyzine HCI (25 rag) or petidine HCI (35 mg). Local anesthesia of the respiratory tract was obtained with topical 2% lidocaine spray. The bronchoscope was positioned in a subsegmental orifice of the right middle lobe. Fifty ml of sterile saline was infused through the bronchoscope into the lung subsegment and aspirated into a container. The wash was repeated 4 times (a total of 200 ml of saline). The lavage fluid was immediately strained through several layers of loose cotton gauze to remove mucus and centrifuged for 10 min at 250 g. The supernatant was used for phospholipid analysis and the aliquot was used for protein determination.

Phospholipid Analysis Lipids were extracted by the method of Bligh and Dyer [9]. Lipid phosphorus was determined by the method of Bartlett [5]. For the analysis of phospholipid composition, individual phospholipids were separated by two-dimensional thin layer chromatography with a 0.25-mm layer of Silica gel G plates prepared with 0.4 M boric acid [21]. The solvent systems used were: chloroform-methanol-

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Table 1. Phospholipid content of bronchoalveolar lavage fluids Phospholipid content (nmol/ml) Normal (n = 8)

IPF (n = 6)

Sarcoidosis (n = 4)

Eosinophilic granuloma (n = 3)

38.2 --- 9.2

26.5 --- 7.4*

29.9 - 8.4

35.9 --- 13.2

IPF: interstitial pulmonary fibrosis. Values are means -+ SD (n). p < 0.05 compared to values for normal.

conc. ammonium hydroxide (70 : 30 : 3 : 2, v/v) for the y dimension and chloroform-methanol-water (65 : 35 : 5, v/v) for the x dimension. Each lipid was identified by cochromatography with known samples of phospholipids isolated from rat liver and lung or prepared enzymatically in our laboratory. After development, spots on the plates were detected by iodine vapor. Individual spots were scraped off and analyzed for lipid phosphorus. The quantitative analysis of molecular species of PC was carried out by high performance liquid chromatography (HPLC) of dinitrobenzoyl derivatives of diacylglycerols (DNB-DG) derived from PC essentially according to the method of Kito et al. [17]. The PC was purified by twodimensional thin layer chromatography as described above. The PC spot on the plates was scraped and eluted from the gels with chloroform-methanol-acetic acid-water (50 : 39 : 1 : 10, v/v) as described by Arvidson [4]. The eluate was washed with 4 N ammonium hydroxide and 50% methanol. The purified PC was treated with phospholipase C from Bacillus cereus (Sigma Chemical Co., St. Louis, MO, USA), and the degradation product was isolated by one-dimensional thin layer chromatography with a solvent system of hexane-ether-acetic acid (50:50: 1, v/v). The !,2-diacylglycerol thus prepared was mixed with 25 mg of dried 3,5-dinitrobenzoyl chloride, and the mixture was dissolved in 0.5 ml of dry pyridine and heated in a sealed vial at 60°C for 10 rain. Then 0.5 ml of water was added and the solution was heated at 60°C for further 10 min. The product was extracted with n-hexane as described by Kito et al. [17]. Samples (DNB-DG) were dried under N~. stream to remove hexane, the residue was then dissolved in acetonitrile, and a 20-50 tzl aliquot of the solution was applied to HPLC. The HPLC was carried out on a BAS liquid chromatographic system equipped with a variable wavelength detector (model UVITEC-100, Nihon Bunko Co., Japan), which was operated at 254 nm in conjection with an integrator (Shimadzu Chromatopac 51A). The DNB-DG was separated by chromatography on a 250 x 4.6 mm Hibar lI column packed with LiChrosorm RP-18 (10 um) (Merck, Darmstadt, FRG). The solvent system was an isocratic solvent, acetonitrile/isopropanol (80 : 20, v/v), pumped at a flow rate of 1 ml/min at room temperature.

Statistics The statistical significance was evaluated using Student's t-test.

Results The phospholipid content per ml of BAL fluid was significantly decreased (p < 0 . 0 5 ) i n I P F c o m p a r e d t o n o r m a l s u b j e c t s ( T a b l e I), w h i l e t h e r e w a s n o d i f f e r ence among sarcoidosis, EG, and normal subjects.

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Table 2. Phospholipid composition of bronchoalveolar Iavage fluids Phospholipid composition (mol%)

Phosphatidylethanolamine Phosphatidylcholine Sphingomyelin Lysophosphatidylcholine Phosphatidylserine Phosphatidylinositol (PI) Phosphatidylglycerol (PG) Bis (monoacylglycero) phosphate Ratio of PG/PI

Normal (n = 8)

IPF (n = 6)

Sarcoidosis (n = 3)

Eosinophilic granuloma (n = 2)

3.5 ± 1.4 76.7 ± 1.9 2.0 -+ 0.4 0.7 - 0.2 1.1 ± 0.4 2.6 ± 0.2 10.8 ± 1.6 2.6 ± 1.1

3.5 75.1 3.7 0.8 2.3 4.7 7.2 2.3

3.t 75.1 ± 2.0 ± 0.9 ± 1.3 2.3 ± 12.4 ± 2.9 ±

3.9 76.6 2.1 0.9 t.8 3.5 8.5 2.7

4.3 ± 0.8

--- 0.9 ± 4.2 ± 1.0"* ± 0.3 + 1.2" ± 1.6"* ± 2.0** + 0.7

1.7 ± 0.7***

0.6 0.4 0.9 0.5 0.4 0.5 1.6 0.8

5.7 -+ 1.5

±1.1 ~- 2.3 -+ 0.2 -+ 0.3 ±0.1 -+0.1 + 0.3 + 1.2

2.5 -+0.1

IPF: Interstitial pulmonary fibrosis. Values are means + SD (n). * p < 0.02 compared to values for normal. ** p < 0.005 compared to values for normal. *** p < 0.001 compared to values for normal. As seen in T a b l e 2, PC was the p r e d o m i n a n t phospholipid that a c c o u n t e d for 76.7% in n o r m a l subjects, 75.1% in I P F , 75. I % in sarcoidosis, and 76.6% in E G . H o w e v e r , there w e r e s o m e differences in the phospholipid c o m p o s i t i o n b e t w e e n I P F and n o r m a l subjects. A significant d e c r e a s e (p < 0.005) in PG was f o u n d in I P F . P G a c c o u n t e d for 10.8% in n o r m a l subjects but for o n l y 7.2% in I P F . In c o n t r a s t , phosphatidylinositol (PI), s p h i n g o m y e l i n , and p h o s p h a t i dylserine w e r e significantly higher in I P F than in n o r m a l subjects. T h e ratio o f P G to PI, w h i c h are k n o w n to be f o r m e d f r o m the same p r e c u r s o r , CDPd i a c y l g l y c e r o l (DG), was significantly d e c r e a s e d in I P F (1.7 - 0.7) in c o n t r a s t to normal subjects (4.3 - 0.8)(p < 0.001). T h e r e was no r e m a r k a b l e c h a n g e in the p h o s p h o l i p i d c o m p o s i t i o n in sarcoidosis and E G . T h e s e findings indicate that the ratio o f P G to PI in B A L fluid m a y be useful in distinguishing these three interstitial lung diseases. T h e H P L C separation profiles o f molecular species o f PC in B A L fluid f r o m n o r m a l subjects and I P F are s h o w n in Figure 1. T w e n t y - o n e p e a k s w e r e detected in the c h r o m a t o g r a m s . T h e molecular species c o n t a i n e d in e a c h p e a k was designated a c c o r d i n g to the p e a k identification as described by I t o h et al. [15] in o u r l a b o r a t o r y . The p r e d o m i n a n t p e a k w a s dipalmitoyl ( 1 6 : 0 / 1 6 : 0 ) species, w h i c h p o s s e s s e s palmitic acid both at the 1- and 2-positions. O t h e r main p e a k s w e r e u n s a t u r a t e d species such as 16 : 0/18 : 2, 16 : 0/16 : 1, a n d 16 : 0/ 18 : 1 species. In Table 3, the relative a m o u n t s o f dipalmitoyl species a n d the 3 u n s a t u r a t e d species w e r e c o m p a r e d in interstitial lung diseases a n d n o r m a l subjects. Dipalmitoyl species was significantly (p < 0.05) d e c r e a s e d in I P F (49.6%) and sarcoidosis (50.0%) c o m p a r e d to n o r m a l subjects (61.3%). T h e value o f dipalmitoyl species in E G s e e m e d to be different b e t w e e n normal

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A le:0/18:0

.,.o,,., ~ ~ 1 1i1l : 0 1 1 8 : 1 B

16:0/16:0

Fig. 1. High performance liquid chromatographic separations of molecular species of phosphatidylcholines of bronchoalveolar lavage fluid from normal subject (A) and interstitial pulmonary fibrosis (B).

18:0/16:1 t6;0/18:2

16:0[18:1 10

20

30

40

50

Retention time (min) subjects and EG, although this was not statistically significant. It is, therefore, likely that a decrease in dipalmitoyl PC is a c o m m o n feature in interstitial lung diseases. In contrast, 16 : 0/16 : 1 and 16 : 0/18 : 2 species seemed to be higher in IPF, sarcoidosis, and E G than in normal subjects, although some of these differences were not significant.

Discussion The present results clearly demonstrated that the phospholipid profiles of B A L fluid are significantly altered in intestinal lung diseases, particularly in IPF. The prominent changes in BAL-phospholipids in IPF are (1) decrease in the phos-

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Table 3. Molecular species composition of phosphatidylcholine of bronchoalveolar lavage fluids Molecular species composition (%)

16:0/18:2 16:0/16:1 16:0/18: I 16:0/16:0 Others

Normal (n = 7)

IPF (n = 6)

Sarcoidosis (n = 4)

Eosinophilic granuloma (n = 2)

8.6 11.4 8.9 61.3

10.9 14.6 7.0 49.6

9.8 14.3 8.9 50.0

10.3 17.4 9.0 50.4

--- 0.4 ± 0.2 ± 2.8 ± 6.5

9.8 ± 3.1

- 2.9 ± 2.4* -+ 3.5 --+ 6.0*

18.2 ± 8.2

--- 2.3 ± 3.4 - 1.3 --- 3.4*

17.0 ± 5.4

+-- 1.9 --- 0.9 - 0.5 ± 1.6

I2.8 + 2.1

IPF: Interstitial pulmonary fibrosis. Values are means - SD (n). p < 0.05 compared to values for normal.

pholipid content, (2) decrease in the PG to PI ratio, and (3) relative decrease in the dipalmitoyl species of PC. It has been reported that saturated PC is decreased in many pulmonary diseases [24, 26, 28]. Dipalmitoyl PC is well known to be a main phospholipid component of pulmonary surfactant, which is synthesized in alveolar Type II cells and secreted into the alveolar space [27]. Therefore, it appears possible that the decrease in saturated PC, predominantly dipalmitoyl species, in BAL fluid reflects some damages in alveolar Type II cells, although it is uncertain whether it reflects functional disturbance in alveolar Type II cells or is the result of quantitative decrease in alveolar Type II cells. PG is well known to be a second characteristic phospholipid in pulmonary surfactant [27]. Dipalmitoyl PC is contained not only in alveolar Type II cells but also in other pulmonary cells, while appreciable amounts of PG are contained in alveolar Type II cells but only trace amounts in other cells [16, 19]. Therefore, change in the PG content in BAL fluid appears to reflect more precisely the damages in alveolar Type II cells than does the dipalmitoyl PC content. The present result showed that PG in BAL fluid is significantly decreased in IPF, but not in sarcoidosis. This finding suggests the possibility that there is a qualitative or quantitative difference in alveolar Type II cells between IPF and sarcoidosis. The present result also showed significant decreases in PI, sphingomyelin, and phosphatidylserine in BAL fluid of IPF. These phospholipids are minor components of pulmonary surfactant, and their roles in surfactant function are not clarified. However, it should be noted that the PG to PI ratio is significantly decreased in IPF, although the total amounts of PG and PI in BAL fluid are similar between IPF and the others. Both acidic phospholipids, i.e., PG and PI, are synthesized from a same precursor CDP-DG in microsomes of alveolar Type II cells and secreted into the alveolar space [6]. In this respect, the result observed in this study, i.e., a significant decrease in

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PG and a concomitant increase in PI in IPF, seems to indicate that a switchover in the biosynthesis of both acidic phospholipids from CDP-DG in alveolar Type II cells may occur in IPF. Thus, in this pathological state, PI synthesis from CDP-DG seems to be enhanced, but PG synthesis is suppressed. The mechanism by which the switch-over of PG and PI biosynthesis is regulated remains to be solved. However, it has been reported that a similar switch-over of the acidic phospholipid formation occurs in the lung at fetal periods when the differentiation of alveolar Type II cells becomes active [13]. Hallman and Epstein [12] suggest that serum myoinositol concentration regulates the switchover of the acidic phospholipid biosynthesis in alveolar Type II cells. PI synthesis in the cells is enhanced during fetal periods when serum myoinositol concentration is much higher, but PG synthesis becomes more active than PI synthesis with decreasing myoinositol concentration in serum. It appears possible that such situations may occur in alveolar Type II cells in IPF. On the other hand, morphological changes in IPF have been reported, that is, a hypertrophy of alveolar Type II cells at the early stage of interstitial pneumonitis and decrease in the number of alveolar Type II cells at the late stage of fibrosis with progression of bronchiolization in the alveoli [10]. Further investigations are required to elucidate the mechanism of the decrease in dipalmitoyl PC and PG. Phospholipids in BAL fluids have been analyzed in healthy volunteers [18], alveolar proteinosis [2, 20, 25], adult respiratory syndrome (ARDS) [7, 14], sarcoidosis [8], and bacterial pneumonia [7]. Hallman et al. [14] demonstrated that, in ARDS, phospholipids in BAL fluids were qualitatively different from those in normal controls. PC, PG, and disaturated PC were low, whereas sphingomyelin and phosphatidylserine were prominent. Low PC to sphingomyelin ratio (<2) and low PG (1% or less of total glycerophospholipids) in BAL fluid were always associated with ARDS, and they pointed out the possibility that the synthesis and secretion of pulmonary surfactant is deficient in ARDS. Baughman et al. [8] reported the decrease in the absolute amount of disaturated PC in BAL fluid of patients with sarcoidosis. They suggest that the decrease in disaturated PC in BAL fluid increased the proliferation of lymphocytes in the sarcoid lung, because disaturated PC has been shown to supress lymphocyte proliferation [3]. Characteristics of fatty acids in phospholipids in BAL fluid have been also studied in ARDS and bacterial pneumonia [7]. Furthermore, the finding that large amounts of pulmonary surfactant accumulate in the alveoli of patients with alveolar proteinosis has been shown by several investigators, i.e., large amounts of dipalmitoyl PC [2, 20, 25] and 36 kD surfactant-associated glycoproteins [20]. However, it appears that the PG content in BAL fluid of alveolar proteinosis is somewhat lower than that of normal subjects [20]. Our present study of phospholipids in BAL fluid of interstitial lung diseases showed decreases not only in dipalmitoyl PC but also in PG to PI ratio in IPF. The new parameter of BAL-phospholipids, i.e., PG to PI ratio, should be studied in other lung diseases. Acknowledgment. This study was supported in part by a Research Grant for Interstitial Lung Diseases from The Ministry of Health and Welfare, Japan.

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24. Ryan SF, Liau DF, Loomis-Bell AL, Hashim SA, Redington-Barrett C (1982) Correlation of lung compliance and quantities of surfactant phospholipids after acute alveolar injury from Nnitroso-N-methylurethane in dogs. Am Rev Respir Dis 123:200-204 25. Sahu S, DiAugustine RP, Lynn WS (1976) Lipids found in pu[monary lavage of patients with alveolar proteinosis and in rabbit lung lamellar organelles. Am Rev Respir Dis 114:177-185 26. Shelly SA, Kovacevic M, Paciga JE, Balls JU (1979) Sequential changes of surfactant phosphatidylcho[ine in hyaline-membrane disease of the newborn. N Engi J Med 300:112-116 27. van Golde LMG (1986) Biochemical aspects of the pulmonary surfactant system. Lipids and membranes: Past, present and future. Op den kamp JAF, Roelofsen B, Wirtz KWA (eds) Elsevier Sci Publisher, Amsterdam, pp. 287-305 28. Von Wichert P, Kohl FV (1977) Decreased dipalmitoyl lecithin content found in lung specimens from patients with so-called shock-lung. Intensive Care Medicine 3:27-30 Accepted for publication: 10 March 1988