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ANALYSIS OF ALVEOLAR LIPIDS 95:1 i'-HííKÜi IN SILICOTIC RATS

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EUR 5123 e

THIN LAYER AND GAS-CHROMATOGRAPHIC ANALYSIS

OF ALVEOLAR LIPIDS IN SILICOTIC RATS

by R. BATTI, Β. ARNOUX, G. LAMY and R. MASSE

Association: European Atomic Energy Community — EURATOM

Commissariat à l'Énergie Atomique — CEA

Work performed at the Commissariat à l'Énergie Atomique

Fontenay-aux-Roses (France)

Association No. 100-72-1 BIAF

Luxembourg, April 1974 — 18 Pages — 2 Figures — B.Fr. 40.—

The incorporation of 1 4C Acetate into alveolar lipids has been studied in

normal and silicotic rats. The surfactant was obtained by endobronchial saline

lavage and the lipids were quantitated by thin-layer and gas-chromatography.

The rate of incorporation of ' 4 C Acetate in comparison to control rats

decreased in silicotic rats in spite of a high deposit of lipids in the alveoli.

EUR 5123 e

THIN LAYER AND GAS-CHROMATOGRAPHIC ANALYSIS

OF ALVEOLAR LIPIDS IN SILICOTIC RATS

by R. BATTI, Β. ARNOUX, G. LAMY and R. MASSE

Association: European Atomic Energy Community — EURATOM

Commissariat à l'Énergie Atomique — CEA

Work performed at the Commissariat à l'Énergie Atomique

Fontenay-aux-Roses (France)

Association No. 100-72-1 BIAF

Luxembourg, April 1974 — 18 Pages — 2 Figures — B.Fr. 40.—

The incorporation of '*C Acetate into alveolar lipids has been studied in

normal and silicotic rats. The surfactant was obtained by endobronchial saline

lavage and the lipids were quantitated by thin-layer and gas-chromatography.

The rate of incorporation of 14C Acetate in comparison to control rats

decreased in silicotic rats in spite of a high deposit of lipids in the alveoli.

EUR 5123 e

THIN LAYER AND GAS-CHROMATOGRAPHIC ANALYSIS

OF ALVEOLAR LIPIDS IN SILICOTIC RATS

by R. BATTI, Β. ARNOUX, G. LAMY and R. MASSE

Association: European Atomic Energy Community — EURATOM

Commissariat à l'Énergie Atomique — CEA

Work performed at the Commissariat à l'Énergie Atomique

Fontenay-aux-Roses (France)

Association No. 100-72-1 BIAF

Luxembourg, April 1974 — 18 Pages — 2 Figures — B.Fr. 40.—

The incorporation of ' 4 C Acetate into alveolar lipids has been studied in

normal and silicotic rats. The surfactant was obtained by endobronchial saline

lavage and the lipids were quantitated by thin-layer and gas-chromatography.

The rate of incorporation of 1 4C Acetate in comparison to control rats

decreased in silicotic rats in spile of a high deposit of lipids in the alveoli.

EUR 5123 e

Association

European Atomic Energy Community - EURATOM

Commissariat à l 'Énergie Atomique - C.E.A.

THIN LAYER AND GAS-CHROMATOGRAPHIC

ANALYSIS OF ALVEOLAR LIPIDS

IN SILICOTIC RATS

by

R. BATTI, Β. ARNOUX, G. LAMY and R. MASSE

1974

Association No. 100 - 72 - 1 BIAF

ABSTRACT

The incorporation of 14C Acetate into alveolar lipids has been studied in normal and silicotic rats. The surfactant was obtained by endobronchial saline lavage and the lipids were quantitated by thin-layer and gas-chromatography.

The rate of incorporation of 14C Acetate in comparison to control rats decreased in silicotic rats in spite of a high deposit of lipids in the alveoli.

KEY WORDS

LIPIDS GAS CHROMATOGRAPHY THIN-LAYER CHROMATOGRAPHY RATS LUNGS CARBO 14 COMPOUNDS ACETATES PNEUMOCONIOSIS UPTAKE TRACER TECHNIQUES BIOCHEMISTRY

— 3 —

C O N T E N T S

1. - Introduction . 5

2 . - Mater ia l s and Methods. 5

2 . 1 . An ima l s .

2 . 2 . l^C Acetate Incorporat ion. 6

2 . 3 . P repara t ion of Total Lipid Samples . 6

3 . - Lipid Analysis . 6

3 . 1 . Thin-Layer Chromatography ( T . L . C . ) . 6 3 .2 . Gas-Liquid Chromatography . 6

3 . 3 . Radioactive M e a s u r e m e n t s . 9

4 . - Resul ts and Discuss ion . 9

5 . - References . L

— δ -

1 . - Introduction

It has been shown by Baily (1963) that the administration of silica

at toxic dose leads to an increase in alveolar lipids. This increase

has also been observed in various toxic dustingsand especially

after Plutonium oxide administration, Masse (1971).

This anomaly is observed in the rat during the blockage of the

physiological alveolar clearance, Nenot (1971), Le Bouffant (1971).

It might either express a decrease in the turnover of alveolar lipids,

or it might be primitive and therefore condition the cellular motili­

ty which converges to the cellular mobilization.

Alveolar lipids are in great part produced by Pneumocyte II

Buckingham et al (1966), but alveolar macrophages which are

numerous in silicotic intoxication have also a synthetic activity,

Pfleger et al (1969).

In the present work, we compare the lipid composition of the alveoli

in normal and silicotic r a t s , with the tendency in alveolar synthesis

from l^c acetate introduced in the alveoli.

2. - Materials and Methods

2 . 1 . Animals

Ten male OFE rats kept in an heteroxenic state and weigh­

ing 350 g before being sacrified were used. After anesthesia

with Nembutal experimental silicosis was induced by an intra­

tracheal injection of 40 mg of Ni silica in 0. 4 ml of a sterile

physiological saline solution. 95% of the injected particles

6 —

were l e s s than 5 ,u in s i z e . The r a t s were kept in a conven­

tional environment for two months and compared to ten con­

t ro l s .

2 . 2 . 1 4 C Acetate Incorporat ion

14 150 ,uCi C Acetate having 50 - 100 m C i / m M were deeply

injected into the t r a c h e a . The react ion was blocked 15 minutes

after the injection of the labelled ace ta te by an in t r a t r achea l

injection of 5 ml of a solution 10 M of sodium ace t a t e .

2 . 3 . P repa ra t ion of Total Lipid Samples

Ext rac t ion of the a lveolar content was performed by lung

washing according to the method of Bra in (1968). A solution

of 50 ml was col lected. This solution contained: in t raa lveo la r

ce l l s , lipidie mice l lae showing the pic ture of mal ta c r o s s

when observed with c rossed niçois and centrifuging with the

ce l l s , free l ipids or combined with prote ins Pasqu ie r (1970).

The lipids were extracted by the Folch (1957) p r o c e d u r e .

Total l ipids were determined by weight . F o r the determinat ion

of phospholipids, the lipidie phosphorus was de termined by

the method of F iske and Subbarow, with about 3. 8% mean

concentrat ion of phosphorus in the phospholipids of the lung.

3 . - Lipid Analysis

3 . 1 . Thin - Layer Chromatography ( T . L . C . )

Lipid analys is was c a r r i e d out by th in - layer chromatography

— 7

without any p r io r separa t ion of neu t ra l l ipids and phospho­

lipids .

All th in- layer chromatographic p rocedures were authenticat­

ed with lipid s tandards ( > 98% pure) of known composi t ion.

F o r the separat ion of the l ip ids , a two dimensional T . L . C ,

was performed with a 250 ,u- thick layer of s i l ica gel HR:

(35 g Merck si l ica in 70 ml H O ) . Resolution of the l ipids

was attained in the following solvent s y s t e m s :

st (1 Development solvent 1 Chloroform: Methanol: water

( )

,nd \2 Development solvent 3

75 : 25 : 4, /v

Pentane: Diethyloxide: F o r m i c

Acid

90 : 30 : 1, V / v

II

st 1 Development solvent 2

2 Development solvent 3

Tetrahydrofuran: dimethoxy

methane: methanol: ammonia

10 : 6 : 4, 1 V / v

Lipid a r e a s were identified by exposing s tandards and sample

a r e a s to iodine vapour s . Individual components were visual iz­

ed by exposure to the following r eagen t s :

- 50% Sulfuric Acid for Choles terol

- Anthrone for Glycolipids

- Schiff-periodate (Niçois 1964) for d ig lycer ides , monogly-

ce r ide s and for identification of phosphatidylglycerol

(Pfleger 1969) in the phosphatidyl dimethanolamine spo t .

- Draggendorf for phosphatidylcholine and lipophosphatidyl-

choline.

Figure 1 and 2 reproduce the thin-layer chromatograms of

the lipid separation.

3.2. Gas-Liquid Chromatography

The lipids were refluxed in methanolic KOH for 2 hours to

transmethylate the fatty-acyl groups.

The free fatty acids were methylated with diazomethane ac­

cording to the method of Schlenk, Gellerman (1960). The

methyl es ters of fatty acids were analyzed on a Perkin

Elmer (Model 900) gas Chromatograph using stainless steel

colums (6 ft χ 1/8 in) packed with 10% ethylene glycol succi­

nate methyl silicone polymer (EGSSX) coated on chromosorb

W(H P) 80/100 Mesh. Columns were prepared and condition­

ed according to the procedure of Wood and Snyder (1966).

The columns were maintained at 170 + 0.1 e C , the dual

hydrogen flame detectors at 2 90°C and the flash heater at

270° C . The flow rates for the FID detectors were air

400 ml/min and hydrogen 42 ml/min. The flow rate of helium

car r ie r gas was 20 ml/min.

A 2. 5 mV, 250 mm strip-chart recorder was used at a chart

speed of 10 mm/min. The fatty acid methyl es ters from the

biological samples were identified by comparing their reten­

tion time with those of known standards. Percentage compo­

sition was determined by half-height analysis of the chroma-

— 9

tographic peaks .

3 . 3 . Radioactive Measurements

l^C radioact ivi ty was measu red in the following scint i l la tor :

POPOP : 0. 3 g - PRO : 5 g - Toluene: 11 with a liquid sc in­

ti l lat ion counter model SL 40 Inter technique. The a r e a s of

the identified spots were scrapped and analyzed d i rec t ly

without any p r io r ex t rac t ion . The radioact ivi ty was m e a s u r ­

ed by desintegrat ion per minutes , and the r e su l t s were com­

pared af te rwards .

4 . - Resul t s and Discussion

The r e s u l t s reproduce the mean values of the pool of ten an imals

in each ba tch .

Table 1 - Composition of Lipids

Total l ipids

Phospholipids

M Phospholipids ° Total l ipids

Control

5. 7 mg

4. 6 mg

80.7%

Silicotic

7 6. 6 mg

17 . 4 mg

22.5% . j

— 10 —

14 Table 2 - Rate of C Acetate

Incorporat ion into Lipids

Control

2%

Silicotic

0 . 1 %

In the solvent sys t em 3-1 phosphatidyl e thanolamine, phosphatidyl

g lycerol and phosphatidyl inositol a r e par t ia l ly reso lved and the

scraping of the sample a r e a s is therefore difficult. The whole r e ­

presen t 24.4% °f the incorporat ion of 1 4 C Acetate into the phospho­

l ip ids . In the solvent sys tem 3-2, these th ree components a r e

completely r e so lved . The radioact ivi ty of the sc raped a r e a s c o r ­

respond to 28.4% of that of phospholipids. On the other hand, ly so -

phosphatidyl chol ines , sphingolipids and phosphatidyl choline a r e

not resolved in this solvent s y s t e m .

The solvent sys tem 3-1 gives on the con t ra ry a good reso lu t ion .

The ra t e of incorporat ion of the 1 4 C Acetate into Lipids is shown

in table 3 whic^ r e p r e s e n t the values obtained in the two chroma -

tog rams for the highly resolved spo t s .

— 11

Table 3 - Rate of incorporation of Radioactive Percursor

into Lipids

Lipids

Choles terol E s t e r

Tr ig lyce r ides

Fa t ty Acids

Rf 0.62 unidentified

Choles te ro l

Monoglycerides

Dyglycerides

Glycolipids

Phosphatidyl ethanolamine

Phosphatidyl g lycerol ) (

Phosphatidyl d imethanola- ) mine ) Phosphatidyl choline

Phosphatidyl inositol

Sphingolipids

Lysophosphatidyl cholines

Control

5.1

6 .6

3 .7

11.7

8 .3

7 . 2

7 . 1

6 .6

5 .2

4 . 8

19.2

5 .6

1.8

3 . 4

Silicotic

3 . 6

13.2

33.6

2 . 2

2 . 5

3 .2

14.2

7 . 1

5 . 4

5 . 3

5 .7

1.4

2. 9

1.0

The fatty acid composition of the lipid from pulmonary surfactant

is shown in table 4.

— 12

Table 4 - Fa t ty Acid Composition of Pulmonary (x) Surfactant Percen tage of Total Fat ty Acids

F a t t y A c i d s

14:0

16:0

16:1

18:0

18:1

18:2

20:4

% S a t u r a t e d in S u r f a c t a n t

% U n s a t u r a t e d in Su r f ac t an t

C o n t r o l

7 . 2 2

6 4 . 5 1

8 .33

9 .68

6 .62

3 .62

+

8 1 . 4 1

1 8 . 5 7

S i l i co t i c

1 5 . 90

5 6 . 0 3

1 0 . 1 3

5 . 0 9

8 .35

4 . 4 7

+

7 7 . 0 2

2 2 . 95

The number before the colon r e p r e s e n t s the number of carbon

a t o m s .

The number after the colon r e p r e s e n t s the number of double bonds

+ Indicates the p resence of a component in amounts l e s s than 1%.

(x) Values a r e mean percentage from four samples

13

The a n a l y s i s of the a l v e o l a r l ip id in the s i l i c o t i c and in the c o n t r o l

r a t s h o w s a m a r k e d d i f f e r e n c e in c o m p o s i t i o n though a s l igh t v a ­

r i a t i o n i s o b s e r v e d in the fa t ty ac id c o m p o s i t i o n . N e v e r t h e l e s s a

h igh i n c r e a s e of the s h o r t cha in fa t ty a c i d i s o b s e r v e d in the s i l i ­

c o t i c r a t .

We o b s e r v e in the s y s t e m u s e d to e v i d e n c e the s y n t h e s i s of a l v e o l a r

l i p i d s :

14 1) a low i n c o r p o r a t i o n of C a c e t a t e in to l i p i d s ;

2) the i m p o r t a n c e of the pool of l a b e l l e d f r e e fa t ty a c i d s ;

3) the e v i d e n t d e c r e a s e of the i n c o r p o r a t i o n of t h e s e fat ty a c i d s

in the p h o s p h a t i d y l c h o l i n e s .

A l though the p r e c e e d i n g d o e s not r e p r e s e n t the p h y s i o l o g i c a l p a t h ­

w a y t h r o u g h wh ich the a l v e o l a r l i p i d s a r e r e n e w e d s i n c e the p r e ­

c u r s o r s a r e i n t r o d u c e d via the b lood s t r e a m , we o b s e r v e t h a t the 14 i n c o r p o r a t i o n of C a c e t a t e i s a s fo l l ows :

N e u t r a l L i p i d s

C o n t r o l

45%

S i l i co t i c

7 1 %

of the a c t i v i t y found in the c h r o m a t o g r a m .

If t h i s i n c o r p o r a t i o n d o e s not r e f l e c t the c o m p o s i t i o n of the a l v e o l a r

l i p i d s for the c o n t r o l r a t

14

Control

Composition

Incorporat ion

Neutra l Lipids

19 .3%

45%

for the Silicotic r a t on the con t r a ry the values a r e much c lose r

Silicotic

Composition

Incorporat ion

Neutra l Lipids

77 .5%

71%

this would mean that the l ipids sec re ted by Pneumocytes II unde r ­

go an a lveolar turn over which modifies the ini t ial composit ion of

the surfactant .

In this medium of low metabolic activi ty but with prolonged effect

due to a lveolar s t a s i s we can p r e s u m e that the enzymatic equipment

of lengthening or condensation of the fatty ac ids a s well a s the s t a ­

ges of t r ans fe r Diglycer ides CDP choline a r e in par t deficient and

consequently would explain the dec rea se of leci thin and the low in­

c r e a s e of g lycer ides and fatty a c i d s . The pathway of synthesis by

methylat ion of the lysolecithin does not appear to be a major factor ,

as the re la t ion Leci thins r e m a i n s a lmos t constant in both Lysoleci thins

control and si l icotic r a t s .

5. - References

B A I L Y P . , KILROE-SMITH T . A . , HARING TON J . S . , 1963. Some

— 15 —

lipid const i tuents of no rma l and quar tz ­dus ted guinea pig lungs .

Nature 198, 856­857.

BRAIN J . D . , FRANK N . R . , 1968. The re la t ion of age to the

number s of lung free ce l l s , lung weight and body weight in r a t s .

Journal of Gerontology_2_3^ 58­62.

BUCKINGHAM S. , HEINEMAN Η. O ., SOMMERS S . C , McNARY

W . F . , 1966. Phospholipid synthesis in the la rge pulmonary a lve ­

olar ce l l . Its re la t ion to lung su r fac tan t s . Amer ican Journal of

Pathology 48, 6 1027­1041.

F O L C H J . , L E E S M . , SLOANE STANLEY G . Η . , 1957. A s i m p l e

method for the isolation and purification of total l ipids from animal

t i s s u e s . Journal of Biol . Chem. 226, 497­509.

LE BOUFFANT L . , 1971. Influence de la na ture des pous s i è r e s

et de la charge pulmonaire su r l ' épura t ion .

In: Inhaled pa r t i c l e s III WALTON W. H.. Ed. Unwin B r o t h e r s , Old

Woking, 227­237.

MASSE R . , 1971. Etude cytologique comparée de l ' influence du

plutonium et de la s i l ice inhalés sur le compor tement du m a c r o ­

phage a lvéo l a i r e . In: Inhaled pa r t i c l e s III, WALTON W . H . E d . ,

Unwin B r o t h e r s , Old Woking, 247­259.

NENOT J . C . , 1971. Etude de l ' influence de l ' i r r ad ia t ion sur l 'épu­

ra t ion pu lmona i re . In : Inhaled pa r t i c l e s III, WALTON W . H . E d . ,

Unwin B r o t h e r s , Old Woking, 239­246.

N I C O L S B . M . , 1964. New biochemical separa t ion . Van Nostrand,

London, 231.

16

PASQUIERCH. , LEFEBVRE J . C . , E H R A R T J . C , 1971. Aspect

biochimique de la rétent ion a lvéola i re p récoce des a é ro so l s l iqui ­

des de t e r r e s r a r e s . In : Inhaled pa r t i c l e s III, WALTON W . H . E d . ,

Unwin B r o t h e r s , Old Woking, 301-309.

PFLEGER R. C , THOMAS G . , 1968-196 9. F i s s ion products . In­

halation p r o g r a m , Lovelace Foundation, Annual Report LF 41, 153.

SCHLENK H . , GELLERMAN J . L . , 1960. Es ter i f ica t ion of fatty

ac ids with diazomethane on a smal l s c a l e . Anal . Chem. 32̂ 1412-

1414.

WOOD R . , SNYDER F . , 1966. Gas - liquid chromatography analys is

of long chain i somer i c g lycer monoe thers .L ip ids 1 62-72.

— 17 —

LIST OF FIGURES

Figure 1 Two-dimensional th in- layer ch roma togram on s i l ica

gel HR (250 ,u thick) of pulmonary surfactant l ipids

from sil icotic ra t in the solvent sys tem 3 - 1 .

F igure 2 Two-dimensional th in- layer ch roma togram on s i l ica

gel HR (250 ,u thick) of pulmonary surfactant l ipids

from si l icotic r a t in the solvent sys tem 3-2.

— 18

TG

I

E.Ch

Soivani

1 —

Salvant

3 τ

f Ch

DG

9 MG G l

PE SP D»pot

PG PC LPC

Figure 1 Two­dimensional thin­layer chromatogram on silica

gel HR (250 ,u thick) of pulmonary surfactant lipids

from silicotic rat in the solvent system 3­1 .

E.Ch

r» TG

Sol von I

3 ♦ Soi vont

— 2

AGNE

J Ch

DG

PC SP LPC

MG GL PG

Figure 2 Two­dimensional thin­layer chromatogram on silica

gel HR (250 ,u thick) of pulmonary surfactant lipids

from silicotic rat in the solvent system 3­2.

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