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Title Experimental Study on Pulmonary Surface Tension in the Normal andPneumonic Lung

Author(s) Kimura, Namiki

Citation Acta Medica Nagasakiensia. 1972, 16(3-4), p.124-138

Issue Date 1972-03-25

URL http://hdl.handle.net/10069/17444

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Acta med. Nagasaki 16 : 124-138

Experimental　 Study　 on　 Pulmonary　 SurfaCe

Tension in the Normal and Pneumonic Lung

Namiki KIMURA *

Second Department of Internal Medicine, Nagasaki University School of Medicine ,

Nagasaki, Japan,

Received for publication, February 15, 1972

The differences of the surface tension between in normal lung and in pneumonic was investigated from the point of lipid metabolism.

Minimal surface tension value was high in pueumonic lung. In the washing extract phospholipid, cholesterol and triglyceride increased in pneu-

monic group and especially the increase of cholesterol and triglyceride was predominant. In the mincing extracts lipid content had no significant diffe-

rence in both groups. Phosphatidylcholine and phosphatidylethanolamine of surfactant obtained from the pneumonic group decreased slightly or lysoleci-

thin and sphingomyelin increased reversely. In the fatty acid composition of phosphatidylcholine of surfactant, palmitic and palmitoleic acid increased

and linoleic acid decreased in pneumonic group. Its composition of triglyce- ride had the decrease of palmitic acid and the increase of oleic and linoleic

acid. The incorporation of CDP-choline-1-2-14C into the lung, the washing

extract and the liver was investigated in normal and pneumonic rabbit. The incorporation into the washing extract was dominant but incorporation into the liver was low. In pneumonic lung the time course of incorporation was

similar to that of normal and its activity was lower than normal. These results indicated that the lung had active lipid metabolism, and

that the pneumonic lung was low in surface activity and its genesis was due to the damaged lipid metabolism and contamination of serum.

INTRODUCTION

Recently the study on the pulmonary surface tension has been reported by many investigators12). The tracheal washing extracts and mincing extracts have been used as materials.

FUJIWARA et a13)4)5)6) reported that a major component of pulmonary

surfactants was dipalmitoyllecithin. In many cases of pneumonia non-

*木 村 南 樹

124

1972 PULMONARY SURFACE TENSION 125 obstructive atelectasis has been recognized and various inferences have been considered as its genesis, but the conclusion has not been establi-shed .

BURBANK7) considered that a genesis of non-obstructive atelectasis based on the decrea-~5e of the radii of the air sacs induced by the weakness of respiratory muscles or paralysis of diaphragrnatic nerve.

SUTNICK8) expected that an anti-pulmonary surfactants was in the pneumonic focus. On the other hand, YOSHIDA9) reported that in the pneumonic exsudate there was some material and pulmonary surface

activity was made low by its material. . In this paper the mechanism of the changed surface tension in the

pneumonic lung was studied from the point of lipid metabolism.

MATERIALS AND METHODS

In order to confirm the condition of mincing method the following preliminary experiment was done. Three or 2.5 gr of the lung tissues were minced, and then were stirred for 5 minutes, 15 and 30 mirlutes. This preliminary experiment revealed that it was the best in the mincing method to mince 3.0 gr of the lung tissues and t.o stirre for 5 minutes .

Healthy rabbits of both sexes maintained on normal rabbit diet and

weighing from 2.0 to 2.5 kg were used. These animals were divided-into two groups, i.e normal and pneumonic group.

In one group _ the _infusion of diplococcus pneumoniae into the trachea was performed and then this group was used as the pneumonic group at 48 hours after the administration of diplococcus. The other group was without any performances and was used as normal.

These two groups were starved for 24 hours, then anesthetized-with 35 mg/kg of pento-barbital sodium and sacrificed by the exsan-

The entire lung was removed for the study. The lung was .perfu-_ sed from the pulmonary arter,y with 100 to 150 ml of saline. On the other hand, according to the mincing method 3.0 gr of left lobe was stirred for 5 minutes by a magnetic stirrer and then was filtered with

a gauze. The tracheal washing extract and mincing extract were adjusted to become 50 ml in volume by the addition of saline, and then were instilled into the teflon trough of the modified Langmuir Wilhelmy surface film balance (Acoma Wilhelmy) for the measurement of surface tension.

After the first surface aging for 30 minutes the surface was alter-natively compressed to one-fith of its area, and reexpanded over 2.5 minutes cycles. A determination was completed when two successive surface area diagram were accomplished, and recorded on X-Y

126 N . KIMURA Vol. 16

recorder .

Stability indices were calculated to the formula provided by CLEM-ENTlo) et al.

S= 2 (r max r mm) / (r max + r mm) The tracheal washing and the mincing extract in the teflon trough

were completely decanted in the flask for the lyophilization after the

measurement of surface tension. Extraction of its lipids was then performed according to the

method of FOLCH and LEES11) . Lung tissue sample weighing 2.5 gr of the perfused right lobe was homogenized in glass homogenizer and used for lipid extraction as mentioned above.

Lipid content of the lung tissue, the tracheal washing extract and the mincing extract was determined by the following methods.

The total amount of lipid was measured by gravimetory, phospho-rus content by the method of ALLENl2) , total cholesterol by the method of ZAK13) and HENLY14), and triglyceride by the method of VAN-HANDEL and ZILVERSMIT15) . Phospholipids were fractionated by the method of WAGNER16) , neutral lipid was fractionated by the method of VOGEL17) and phosphorus content of their subfractions was determined by the micro-quantitative method of NoJIMA18). Fatty acid residue of them was methyl-esterified by the method of STOFFEL19) and RADlN20), and then determined by gas-1iquid chromatography (Shimazu Gas Chromatograph GC-IB). According to the method of OKAMOT021) and DlNESH22) , it was studied that the dilution curve of phosphatidylcholine extracted from

the tracheal washing extract and lung tissue, pure dipalmitoyllecithin , pure fatty acids (saturated and unsaturated), pure triglyceride and pure cholesterol .

In order to study the incorporation of CDP-choline-1-2-14C into the lung tissue, the liver tissue and the tracheal washing extract in normal and pneumonic rabbits, 100 /h Ci of CDP-choline-1-2-14C was injected intravenously into each another rabbit of both groups and sacrificed by exsanguination, at 10 minutes, 6, 12 and 24 hours after the administration of isotope.

Total lipid, total phospholipid and phospholipid subfraction of the lung tissue, the liver tissue and the tracheal washing extract were measured by the same method mentioned above.

Phospholipid and non-phospholipid were separated by column chro-matography and futhermore phospholipid was fractionated by thin layer chrmoatography. The radio-activity of total phospholipid and phospha-tidylcholine was measured by liquid scintillation spectrometer (Packard Co) .

1972 PULMONARY SURFACE TENSION 1 27

RESULTS

The content of phospholipid, cholesterol and triglyceride in normal rabbit serum were 100.9~21.3 mg/dl, 71.1~15.8m g/dl and 49.8:~23.3 mg/dl respectively. (Table 1)

In phospholipid subfractions, phosphatidylcholine was 76.6~3.2 ~~ , lysolecithin 16.1+_2.8 ~~, sphingomyelin 4.2~2.4 ~ , and phosphatidyle-thanolamine 2.8il.O % . (Table 2)

The fatty acid composition of phosphatidylcholine and triglyceride in

the rabbit serum was shown in Table 3. In normal group, the mean maximal and minimal surface tension

value and stability index of the tracheal washing extract were 38.8~5.3,

2.6+_1.9 dynes/cm and 1.74~0.16, anf those of the mincing extract were 46.7 +_3.4, 5.9~1:2.3 dynes/cm and I .55~0.14 respectively.

In the pneumonic group, the mean maximal and minimal surface tension value of the tracheal washing extract were 45.2:k4.3 and 9.1~ 3.5 dynes/cm, and those of the mincing extract were 43.3~3.5 and 14.8+_4.6 dyneslcm, and stability index was 1.34:~0.22 in the tracheal

washing e･xtract and I .O0+_0.27 in the mincing extract. (Table 4)

Table 1. Li pid Fraction of Rabbit Serum

Total Amounts (mg/dl)

Total Cholesterol

Triglyceride

Phospholi pid

Total Amounts (~)

Total Cholesterol

Triglyceride

Phospholipid

Normal

221 . 8 J,,_._.-45 . 6

71 . I ~ 15 . 8

49 . 8 ~J- 23 . 3

100 . 9 ~J- 21 . 3

100

32.8-__-'- 7.0

21.6~ 7.5

45.6+ 4.0

(14)

Pneumonie

299 . 7 ~ 13 .

37.5~ 7.

15.6i: 4.

140.0~ 8.

1 OO

37.5+_ 7.

15.6:f: 4.

46.7d: 2.

O

2

9

7

2

9

8

(3)

() No of materials

Table 2. Phospholipid Subfraction of Normal Rabbit Serum (mean of 14cases)

Total Amounts(%)

Phosphatidylcholine

Lysolecithin

S phingomyelin

Phosphatidylethanolamine

1 OO

76.6~3.2

16.1~2.8

4.2+_2.4

2.8~1.0

128 N. KIMURA Vol. 16

In 2.5 gr of the wet lung tissue, the sum of the phospholipid, cholesterol and triglyceride was 43.2:k6.7 mg in normal group, and 37.4+_4.9 mg in the pneumonic group. Cholesterol was 8.3~:1.2 mg in normal group and 7.1~:1.1 mg in the pneumonic group. Triglyceride was 1.7~1.0 mg in normal group, and 2.4~1.2 mg in the pneumonic grou p .

In the tracheal washing extract of the normal group, the sum of phos-

pholipid, cholesterol and triglyceride was 8.0~2.6 mg, phospholipid was

7.1~2.5 mg, cholesterol was 0.8~0.3 mg and triglyceride was 0.14~ 0.03 mg. In the tracheal washing extract of pneumonic group, these

Table 3. Fatty Acid Composition of Phosphatidylcholine

in Rabbit Serum

and Triglyceride

12 : O

14 : O

16 : O

16 : 1

18 : O

18 : 1

18 : 2

18 : 3

20 : 4

undetermined

S/ U

Phos phatidylcholine

Normal

0.4'T_0.1

0.4~:0.1

27. OJ_-'- I . 1

2.6:L-L0.9

21.3~3.0 12.3-J-2.2

29. I J-'.__1 . 3

0.9J_J-'0.3

2.6J_,_-.0.3

3. I J'___-'O. 5

1.04~J-O 04

(4)

Pneumoine

trace

0.4H.=.-･0.1

24 . 3~+ 2. O

1.7~J-O 2

22.8~*-1.9

9.1~1.3 34 . 8 ･k I . 8

1.2:1:0.7

3.9~1.3

1.8~0.3

O . 94 :~:O . 05

(4)

Triglyceride

Normal

0.5~0.4 1.4:1:_0.4

39.9J-J~2.7

5.4J_J-1.1

4.2J_J-.1.1

26 . 3 ~:: 3 . 4

15.9:{:2.2

1.6~-'-0.5

1.1J_J!0.6

3.8d:0.7

O. 93~0. 16

(4)

() S/ U

No of materials The ratio of saturated to unsaturated fatty acids

Table 4. The Value of Surface Tension in the Normal and Pneumonic Rabbit Lung

T-max

r-min

S

Normal Group

Washing Extract Mincing Extract

38 .

2.

1.

8 +_5.3

6 ~1.9

74 ~+ O . 16

(8)

46.7 -J~-'-3.

5.9 :1 2.

1 . 55 ~0.

(7)

4

3

14

Pneumonic Group

Washing Extract Mincing Extract

45.2 ~:4.3

9.1 _+_3.5

1.34~J-O 22

(10)

43.3 Jr_3.5

14.8 ~4.6

1 . OO J-~0 . 27

(10)

() T-max T-min

S

No of materials

Maximum surface Minimum surface Stability index

tension

tension

(dynes/cm) (dynes/cm)

1972 PULMONARY SURFACE TENSION 129

were 17.1~7.7 mg, 12.8~6.2 mg, 3.1:~:1.3 mg, and 1.2~0.7 mg res-pectively. In the trachel washing extract of the pneumonic group, the content of cholesterol and triglyceride increased apparently than of

the normal. When the total amount of phospholipid, cholesterol and triglyceride

was slided in scale to 100% , phospholipid occupied 87.9~3.9% in normal

group and 72.6:~7.6~~ in the pneumonic group. In the pneumonic group, its percent of cholesterol and triglyceride increased apparently than those of normal group. (Table 5)

In normal rabbits specific activity of phospholipid of the lung tissue

had a peak at six hours and then gradually decreased until 24 hours, although the peak in the liver tissue was at 12 hours and it was lower

Table 5. Lipid Fraction of Lung Tissue, Washing Extract and Mincing Extract

Total Amounts (mg)

Total Cholesterol

Triglyceride

Phos pholipid

Total Amounts (%) Total Cholesterol

Triglyceride

Phospholi pid

Normal Gr ou p

L. Tissue W. Extract M. Extract

43 2~6 7 8 8.3-J_-'-1.2 O

1.7~1.0 O. 33 2~J-.5 3 7

100

19.4~2.1 9 3.7:~2.1 2 76.8~+2.3 87

(11)

.0~2.6

.8~0.3 14 ~- O . 03

. I J,_.-2. 5

100

.8~3.6

.0~+1.0

.9~3.9

(8)

7

O

O.

6

13

2

84

.1~2.

. 9~0.

13~0. . O -~-'- 2 .

100

.2~1.

.1J!~'~1.

.5~1.

(7)

2

3

06

o

9

3

7

Pneumonic Grou p

L Trssue W. Extract M. Extract

37. 4 ~4 . 9

7.1~~'~1.1

2.4-+_' 1.2

28. O ~3. 1

1 OO

19 . O~: I . 8

6.I~:2.4

74.8~2.3

(8)

17.

3.

12 .

19 .

7.

72 .

1~7.

1~1.

2~0.

8~6. 100

9~6.

4~3.

6~7.

( I O)

7

3

7

2

7

2

6

6.7~.J~2.

1.3J~~' O.

0.5~0. 4.9J~J-2.

1 OO

20 . 3 :~:6 .

8.6J*J~4.

71 . O ~6 .

( I O)

3

5

2

1

7

6

8

W. M.

() Tissue

Extract

Extract

Table 6.

No of materials

2.5 gr of wet lung tissue

Washing Extract from unilateral lobe

Mincing Extract from 3.0 gr of wet lung tissue

Specific Activity of Phospholipid and Phosphatidylcholine (cpm/T)

10

6

12

24

minutes

hours

hours

hours

Lung Tissue

PL PC

P

1 95

5 18

417

372

49

237

151

75

330

842

744

756

88

597

48 4

395

t

Liver Tissue

PL Pc

N P N P

42

234

333

307

48

514

3 34

142

61

314

457

379

76

7 24

517

306

Washing Extract

PL PC

N P N P

29

229

602

698

219

366

/ 49 7

33

320

1097

1263

283

454

/ 479

N P

PL PC

Normal Pneumonie Phsopholipid

Phos phatidylcholine

130 N．KIMURA 70」，16

than　that　of　the　lung　tissue．　Activity　of　the　tracheal　washing　extract

was　lower　than　that　of　the　lung　tissue　at10minutes，6hours　and　was

higher　at　12hours　and　increased　until24hours．

　　　The　time　course　of　the　specific　activity　of　phosphatidylcholine　of

the　lung　tissue，the　liver　tissue　and　the　tracheal　washing　extract　was

700

600

500

400

300

200

100

0

Norma1

　　　　　　　　□／　　　　　　　　，／

　　　　　　　／　　　○

　　　　＼

　　　　　／

　　　　　／　△＿　　　　　　　！／　　　　／－！！！

　　　△〆

　　　口O　　ノ’

　　14　、1／

・1／

丞／

□

／一／一

　　　□　　／／○一〇Lung　Tissue

△一一一一△Liver　Tissue

⊂｝一・一ロWashing　Extract

○

1、一一～

Pneumollia

　　　▲　　　’＼、　　　’　　￥　　　’　　　、

　　　ヨ　　　　　ペ　　　，　　　　　￥，／　　　　　コノペ　　1舅／　＼、

　　 　　　　▲　　’／

　／

／7●ロ、1＼　’　　　　　●　1

’

．／一！一！

￥

／一！一

－■

＼

▲

●

10mins6hrs　　　12hrs 24hrs 10mins6hrs 12hrs 24hrs

Fig．1．　Specific　Activity　of　P・Lipid（cpm／γ）

1400

1200

1000

800

600

400

200

0

Normal Pneu凱onia

口ーノ

，1

　　　　　　　／／

　　　○　み。

　　　　　／／

　　　　／　　　　 △一一隔＿

○　　ノ／

！、〆

一－91口

○

、△

O o　Lung　Tissue

△一一一一一△Liver　Tissue

［：］一・一・一口W　ashing　Extract

　　ズ　，グ

・7

合

／ 口／

2

ノ’

　▲　’　、￥　’　　　　￥￥

　，’●　　￥

1’＼も≧’■一一一一一一甲’／

1■

一＿、一一一、＼　　　●

　　　　　、▲

10mins6｝1rs　　　12hrs

　　　　　　　　　Fig．2，

　　　24hrs　　　　10mins6hrs　　　12hrs

Specific　Activity　of　P．C．（cPm／γ）

24hrs

1972 PULMONARY　SURFACE　TENSION 131

similar　to　that　of　phospholipid　and　its　specfic　activity　of　phosphatidy1－

choline　was　higher　than　of　total　phospholipid．（Table6and　Fig．1，2）．

　　　The　specific　activity　of　phsphatidylcholine　of　the　pneumonic　lung

tissue　was　lower　in　all　time　course　than　that　of　normal　lung　tissue，and

that　of　the　pneumonic　tracheal　washing　extract　was　lower　apparently　at

24hours　after　the　administration　of　isotope，although　at10minutes，6hours，that　of　the　pneumonic　tracheal　washing　extrat　was　higherreversely　than　of　norma1．

　　　The　specific　activity　of　phosphatidylcholine　of　pneumonc　liver　tissue

was　higher　at6hours　than　of　normal　and　that　of　other　time　was　almost

at　same　level　as　norma1．

　　　In　the　mincing　extract　of　normal　group，the　sum　of　phospholipid，

cholesterol　and　triglyceride　was7．1土2．2mg，phospholipid　was6．0土2．O

mg，cholesterol　was　O．9土0．3mg　and　triglyceride　was　O．13土0．06mg．

In　the　pneumonic　group，these　were6．7土2．3mg，4．9±2．1mg，1．3土0．5

mg　and　O．5土0．2mg　respectively．

　　　Phospholipid　occupied84．5±1．7，％in　normal　group　and71．0土6．8％

in　the　pneumonic　group．　Apparently　percent　of　phospholipid　decreasedand　reversely　percent　of　cholesterol　and　triglyceride　increased　in　the

pneumonic　group．（Table5）．

　　　In　phospholipid　subfraction　of　the　wet　lung　tissue，the　difference

between　normal　and　the　pneumonic　group　was　not　significant．　On　the

tracheal　washing　extract　and　the　mincing　extract，phsphatidylcholine

and　phosphatidylethanolamine　of　the　pneumonic　group　decreased　thanthose　of　normal　group．（Table7）．

　　　　In　the　fatty　acid　composition　of　phosphatidylcholine　of　the　wet　lung

tissue，1inoleic　acid　was15．8土0．7％in　normal　group　and20．8土3．7％in　the　pneumonic　group．　In　that　of　the　tracheal　washing　and　mincing

extract，palmitic　and　palmitoleic　acid　decreased　and　reversely　stearic　and

Table7．　Phospholipid　Subfraction

Total　Amounts（％）

　Phosphatidylcholine

　Phosphatidyletha・　　　　　　　　nolamine

　Other　SPots

Normal　Group

L．Tissue　W．Extract　M．Extract

100 100 100

61．9土4．5　84．4土2．1　　85，2土2．5

27．1土4．2　　14．6土3．4　　　13．0土2．1

11．0土3．5

　　　（4）

1．0土0．5

　　（3）

1，8土0．7

　（3）

Pneumonic　GrouP

L．Tissue　W．Extract　M．Extract

100 100 100

60，4土2．7　79．8土5．1　　80，5土4．7

26，4土2，5　　11。1土3。9

13．1土4．6

　　（7）

8，9土1．9

　　（5）

9．8土2．1

9．6土3．1

　（5）

　　　　（）　Noofmaterials

L．Tissue　l　Wet　LungTiSsue

W．Extract　Washing　ExtractM．Extract　Mincing　Extract

132 N．KIMURA 701．16

1inoleic　acid　increased　in　the　pneumonic　group．（Table8）．

　　　　In　the　fatty　acid　composition　of　triglyceride　of　the　tracheal　washing

and　mincing　extract，palmitic　and　arachidonic　acid　apparently　decreased

in　the　pneumonic　group　and　reversely　oleic　and　linoleic　acid　increased．

（Table9）．

Table8．　Fatty　Acid　Composition　of　Phosphatidylcholine

12：014：016；016：118：018：1181218：320：4undetermined

S／U

Normal　Group

L．Tissue　W．Extract　M．Extract

　trace

1．2土0．3

41．2土1．3

6．1土1．0

10．5±2．0

17．7ニヒ1．1

15．8土0．7

1．0土0．2

3．9土0．8

2．5土0．5

1．19土0．12

　　　　（4）

　trace　　　　　　trace

2．5土0．7　　1．6土0．4

54．8土1．9　52．9土3．0

10．5ゴニ1．2　　9．6土2。4

2．4±0．6　　4。0土1，4

17．8±1．0　　18．8土0．9

9．7土2．1　10，2±2．1

0．4土0．3　　0．7土0．1

0，4　　　　　0，2

　1，4土0．5　　1．7土0．5

1．55土0．15　　1．51土0，26

　　　（3）　　　（3）

Pneumonic　Group

lL Tissue　W。Extract　M．Extract

　trace

O．8土0。3

40．8土3，3

6，8土0．9

8．3土0．8

16．4土1．9

20．8土3．7

0。6土0．5

2．5土0．9

2．9土1．5

1．06±0．11

　　　　（5）

　trace

1．9土0．7

50．1土1．0

9．0土0．1

5．1土0．3

17．4土0．8

11．9土2．7

0．9土0．2

0．8土0．6

2．6土1．2

1．43土0，09

　　　（4）

　　trace

1．7土0．3

48．6土2．9

8．2土1．2

6．2土1．9

16．9土1．5

13．1土2．5

1．0土0．5

1．0土0．4

2．8土0．4

1．42土0．11

　　（3）

　　　　　（）

　L．Tissue

W．ExtractM．Extract　　　　　　S／U

No　of　materials

Wet　Lung　TissueWashing　ExtractMincing　ExtractThe　ratio　of　saturated　to　unsaturated　fatty　acids

Table9。　Fatty　Acid　Composition　of　Triglyceride

Normal　Group Pneumonic　Group

　　　12：0

　　　14：0

　　　16：0

　　　16：1

　　　18：0

　　　18：1

　　　18：2

　　　18：3

　　　20：4

undetermined

　　　S／U

Washing　Extract Mincing　Extract

　1．5土0，4

　2．4土0．7

45．8土0．4

　5．7土0．8

　8．5土0．7

　12．3土1．8

　3．8土1．3

　2．3土1．9

　10．8土0．9

　6．4土2．5

1．69土0．21

　　　（3）

2．0土0。1

2．9土0．6

44，8土4．2

5．9土2．6

10．1土2、0

14．1土4．1

5．6土2．0

1．3

7．8±2．9

5．4土2．7

1。72土0．19

　　　　（4）

Washing　Extract Mincing　Extract

1．3土0．7

1．7土1．2

39．1土2．0

6．0±1．9

7．8土0．5

17．8土0．6

11．1土2．8

1．6土0．3

4．6土1．8

8．8土0．6

1．22土0．19

　　　　（3）

　1．4土0．8

　2。3土0637．0土3．8

　7．1土1，8

　6．3土0．9

　19．4±1．2

　14．5±5．0

　1．9±0．4

　3．3土2．2

　6．7土4．3

1．03土0．22

　　　　（3）

（）

S／UNo　of　materialsThe　ratio　of　saturated　to　unsaturated　fatty　acids

1972 PULMONARY SURFACE TENSION 133 No significant difference was recognized between normal and the

pneumonic group in the ratio of saturated to unsaturated fatty acids of phosphatidylcholine of the wet lung tissue, tracheal washing and the mincing extract.

On the other hand, fatty acid composition of triglyceride in the tracheal washing and the mincing extract had significant difference between normal and the pneumonic group. In the pneumonic group, unsaturated fatty acids were rich than those of normal group .

DISCUSSION

It has been discussed which the tracheal washing method or the mincing method is better. In this paper it was revealed that 3.0 gr of the wet lung tissue in the mincing method was adequate in the measu-rement of pulmonary surface tension, and that the sufficient perfusion of

the pulmonary artery made the mincing method as usefull as the tracheal

washing method. BROWN23) compared the tracheal washing method with the mincing

method and he reported that r-min was 5.3 dynes/cm in the tracheal washing method and 7.5 dynes/cm in the mincing mehtod. The content of dipalmitoyllecithin was 4.8 mg% in the mincing extract and 4.0 mg% in the tracheal washing extract. In the fatty acid composition oleic acid increased in the mincing extract than in the tracheal washing extract.

YosHIDA24) studied the relation between the quantity of the lung tissue and its surface tension and he concluded that I .8 gr of the lung

tissue was adequate in the mincing mehtod for the determination of surface tension . It was supported in this paper . According to the method of TIERNEY25) the pulmonary surfactant was obtained because this method was recognized to be the best in the preliminary experi-ment .

It has been commonly believed that there were much more conta-mination in the mincing extract than in the tracheal washing extract. Although this fact was accepted commonly it was thought that the lipid composition of the tracheal washing extract was as same as in the mincing extract if the perfusion of the pulmonaty artery was sufficent.

In pneumonia non-obstructive athelectasis was found and its genesis

was discussed from the various points by BURBANK7), SUTNICK8) and YOSHIDA9)

For the study of the influences of serum on the pulmonary surface tension, TIERNEY25) added from one to six ml of rabbit serum to the mincing extract obtained from normal rabbit lung tissue, and reported that the addition of from one to 2.5 ml of serum to the pulmonary extract had no effect on its pulmonary ;surface tension. He also repor-ted that cholesterol and oleic acid hemmed normal pulmonary surface

tension. By the addition of 40 /hg of cholesterol or I mg of oleic acid ,

r-min became 16 dyneslcm. In pneumonia it was thought that the contamination of serum component would influenced on the pulmonary surface activity.

In order to study the effect of the contamination of serum compo-nent on the pulmonary surface tension, the tracheal washing extract obtained from normal rabbit lung was divided into four equally and then normal rabbit serum was added from one to six ml to them. In each group surface tension was measured, and the contents of lipid and fatty acid composition was analysed.

The addition of from one to three ml of serum had no influence on the minimal surface tension, and the addition of from four to six ml had an influence on minimal surface tension. By the addition of serum maximal surface tension was slightly higher than of the control.

In the fatty acid composition of phsphatidylcholine extracted from the normal surfactant given serum additionaly, palmitic and oleic acid decreased and stearic and linoleic acid increased apparently. That is to say, its pattern became as similar as that of rabbit serum .

On the other hand , in the fatty acid composition of triglyceride palmitic and arachidonic acid decreased and oleic and linoleic acid increased apparently. The fatty acid composition of triglyceride obtai-

ned from the tracheal washing extract of the pneumonic group was similar to that of normal group given from two to four ml of serum.

Judging from these results it was thought that the changes in the pneumonie was due to the mixture of serum induced by conges-tion, and that its mixture of from two to four ml in volume in unilate-ral lung.

72

60

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O O Llnolelc Acid

~<:~-~ Cholesterol

xi X

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10~ 6 10~s lO~ 4 Fig. 3. Tension Concentration Diagram (mM/50cc)

10~3

1972 PULMONARY SURFACE TENSION 135 The lipid content of the tracheal washing extract obtained from the

pneumonic group was similar to that of the washing extracts added from three to four ml of serum.

The length and saturation of fatty acid influenced on the pulmonary surface activity, so the following experiment was done.

According to the method of OKAMOT021) and DlNEsH22) , author described the tension-concentration diagram of pure dipalmitoyllecithin, fatty acids (saturated and unsaturated), triglyceride and phosphatidyl-choline obtained from the tracheal washing extract. And then it was

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o--o Trilinolein

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(mM /50cc ) 10~3

136 N . KIMURA Vol. 16

examined how the contamination of serum in pneumonic group interfe-red the normal pulrnonary surface activity.

Fig. 3. 4. 5, shows the tension-concentration diagram of dipalmi-toyllecithin, fatty acids, triglyceride and cholesterol.

Dipalmitoyllecithin and saturated fatty acid , especially long chain

saturated acids had sufficient pulmonary surface activity. On the other hand, unsaturated fatty acids, triglycerides with unsaturated fatty acids and cholesterol had not sufficient surface activity.

Judging from these results it was concluded that the genesis of the

10w pulmonary surface activity in the pneumonia was due to the increasement of the unsaturated fatty acids of phsphatidylcholine and triglyceride .

It was questionable what induced this increase of the unsaturated fatty acids. A part of this increase was due to serum.

In order to resolve this problem isotope experiment was done . It: has been considered the following ways as the synthesis pathway of phsphatidylcholine, namely (1) KENNEDY'S pathway26) via CDP-choline and D-a, P-diglyceride, (2). BREMER'S phthway27) through the trans-methylation of phosphatidylethanolamine, (3) LAND'S pathway28)29) through lysolecithin and fatty acyl CoA.

FUJIWARA30) reported that the synthesis pathway from CDP-choline and D-a, ~-diglyceride was much more dominant in the normal rabbit lung tissues, the tracheal washing extract and alveolar cell than that from phosphatidylethanolamine, and also reported that the turnover rate

of phosphatidylcholine from CDP-choline and D-a, P-diglyceride was rapid than that from phosphatidylethanolamine. And he said that the specific activity of phsphatidylcholine in the lung tissues was higher tha'n that of the tracheal washing extract until 6 hours since then was 10wer .

In this paper the specific activity of phosphatidylcholine in normal lung was 330 cpm at 1'O minutes after the administration of CDP-choline

- -2-14C, and it was apparently higher than 61 cpm in the liver, 33 cpm in the tracheal washing extract, and its activity arrived at peak of level at 6 hours and its value was 842 cpm since then gradually decreased.

The specific activity of phsphatidylcholine in the tracheal washing extract was higher than that of the lung tissue at 12 hours after the administration of isotope, and gradually increased until 24 hours.

In the liver tissue, the peak of the specific activity of phosphatidyl-

choline was at 12 hours after the administration of isotope.

It was revealed by these results that in the normal lung phosphati-dylcholine was actively synthesized and it was transfered into alveolar lining layer . These results were supported by other investigator ' s ex periments .

On the other hand , the specific activity of phsphatidylcholine of the

pneuminic lung tissues was lower than that of normal lung tissues, and

1972 PULMONARY SURFACE TENSION 137 that Of the tracheal waShing extract waS higher at 10 minuteS and 6 hourS after the adminiStratiOn Of isotope than of the nOrmal waShing extract .

How it waS explained that the activity of the waShing extract Of the

pneumOnic lung waS higher at 10 minuteS and 6 hOurS than Of nOrmal WaShing extract in spite Of the low activity Of the pneumonic lung tiSSueS .

Although thiS initial higher activity of waShing extract of the pneu-

mOnic lung was ObSerVed it WaS accepted that the lipid metabOliSm waS damaged in the pneumonic lung. Although it was thOught that the increaSe Of triglyceride and choleSterol waS induced by the mixiure of

serum due tO the cOngeStiOn, the lipid Synthesis Was aISO Certainly changed. Further experiment of acetate-14C or 1311-albumin muSt COnfirm the mechaniSm of the increaSe of triglyceride and choleSterol, and the invasion of Serum.

ACKNOWLEDGEMENT

The author is grateful to Proffesor Shiro OSAJIMA. Yasutaka KATOH M.D. Kiyoshi KANZAKI M.D. and Atsushi SAITO M.D. for their continuous helpful advice and encouragement .

REFERENCES

1. CLEMENTS, J. A. and TIERNEY. D. F. Handbook of phisiology : Respirationll. Chapter 69. American Physiological Society, Washington, D. C. , 1965.

2. PATTLE, R. E., PHYSIOL. REV. 45. 48, 1965. 3. Tetsuro FUJIWARA, Haruko HIRONO and Tsuneo ARAI(AWA. Tohoku J. exp. Med. , 85.

33, 1965.

4. BRowN, E. S. Aln. J. Physiol. 207. 402, 19'64.

5. KLAUS, M. H., J.A. CLEME)q:TS, and R. J. HAVEL. Proc. Natl. Acad. Sci., US 47. 1858, 1961.

6. MORGAN, J.E., T.N. FlNLEY, and H. FIALKOW. Biockem. Biophys. Acta 106. 403, 1965.

7. BURBANK, B.S.C. CUTLER, and S. SBAR. J. Thoracic a'id Cardiovasc. Surg. 41 . 701, 1961 .

8. SUTNICK, A.1. and L. A. SOLOFF. Al~l~. Inter/~. Med. 60. 39, 1964. 9. YOSHIDA. S.. KITAMURA, S., NAKAO, K. The Japaltese Journa,1 of Thoracic Diseases 4.

350 , 1968 . (Japanese)

10. CLEMENTS, J. A. et al. J. Appl. Physiol., 16. 444, 1961.

11. FoLcH, J., LEES, M. J.Biol. CkerTb. 226. 497, 1957.

12. ALLEN R. J. L. Biochem. J. , 34. 858, 1940. 13. ZAK, B. Aln. J. Cli,~. Path., 27. 583. 1957.

14. HENLEY A. A. A,balyst, 82. 286, 1957.

15. VAN HANDEL E. and ZILVERSMIT D. B. J.Lab. Clil~. Med., 50. 152, 1957. 16. WAGNER H. et al. Biochem Z. 334. 175, 1961. 17. VOGEL, W, C. et al. J. Lipid. Res. 3. 138, 1962.

18. NOJIMA, S. Metabolism a'id Disease, 2. 1023, 1965. (Japanese)

19. STOFFEL, W. et al. Aua~. Chem., 31 . 307, 1959. 20. RADIN, N. S. et al. J.Lipid. Res. I . 250, 1960.

138 N．KIMURA 70Z．16

21．

22．

23．

24．

25．

26，

27．

28．

29．

30．

OKAMoTo，S．イααノ晩d．1〉α8α5α腕．，　14。87，1970．

DINEsH　O．SHAH　and　JAcK　H．Schulman．∫．μP回Re5．8。215，1967，

E．S．BRowN．漉4．Zんorα．22．70，1965．

YosHIDA，S．，KITAMuRA，S．，　ARAI，T．，　and　NAKAo，K．　艶迄Pαπe5θ1ワ診oアαc三〇1）誌e侭e56．　139，　1969。　（Japanese）

D．F．TIERNEY　and　R．P．JoHNsoN．∫．∠丑ρZ．Pゐヅ謡oZ．20．1253．1965。

KENNEDY，E．lP．and　WElss，S．B．1．B♂oZ．C舵㎜．，222．193，1956．

BREMER，J．，FIGARD，P．H．andGREENBERG，D．M．B言ocゐθ㎜．ゐ言o擁ヅ5．477，　1960．

LANDs，W．E．M．∫．B♂oZ．C玩㎜．，235．2233，1960．

LANDs，W・E・M・and　MERKL，LヱB藍oε・C舵冊・，238・898，1963・，Tetsuro　FUJlwARA．Pアooeθdあ85qプ迄Pαπe5θSoσie君ヅノbアB∫oZo8‘cα乙1拓εeヴaoe．，

（Japanese）

ノbμア乃α乙げ

∠〔c彦α．，　43．

1．　5，1969．