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THIN LAYER AND GASCHROMATOGRAPHIC
ANALYSIS OF ALVEOLAR LIPIDS 95:1 i'-HííKÜi IN SILICOTIC RATS
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R. BATTI, Β. ARNOUX, G. LAMY and R. MASSE
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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.
Ali MY
rae wmm l lal l i
1 WW
mۮm \n mm
l ' lW·
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