The incorporation of l-[Me-14C]methionine and [Me-3H]choline into lung phosphatides

BIOCHIMICA

552

ET BIOPHYSICA

ACTA

*BA 55431

THE INCORPORATION

OF

L-

iJfe-‘JC]METHIONINE

AND [Me-3H]CHOLINE

INTO LUNG PHOSPHATIDES

H.

L.

SPITZER,

k;. MORRISON

AND

J. R. NOR&&W

Department of Medicine, Pulmonavy and Gastvoentevology Divisions, The University of Alabama rMedica1 Center, Birmingham, Ala. (U.S.A (Received October z6th, 1967) (Revised manuscript received December

Iqth,

.)

1967)

SUMMARY

A comparison has been made of r.-[Me-Wlmethionine and [Me-3H]chohne incorporation in viva into lung and liver lecithin. The data suggest that the liver is not the source of lung lecithin and that synthesis in S&V occurs via the CDP-choline pathway. The synthesis in Z&JOof lecithin via methylation of phosphatidyl ethanolamine appears to be of only minor importance in lung and probably results from a slow turnover of methionine methyl in this tissue. Using L-[Me-Wlmethionine it has been shown that the phospholipid tentatively identified as phosphatid~~l-~,Ndimethylethanolamine in lung is probably an intermediate in the stepwise methylation of phosphatidylethanolamine. The finding of significant [Me-3H]choline activity in lung and liver phosphatidyl-N,N-dimethylethanolamine reconfirms the original observation that betaine is an important source of a methyl group in the methylation of phosphatidylethanolamine.

INTRODUCTION

A number of investigators have reported that up to 594 of the phospholipids of lung tissue and pulmonary surfactant lipoprotein is phosphatidyl-N,N-dimethylethanolaminel~z. MORGAN, PINLEY AND FIALKOW’ found that the phosphatidyl-N,~~dimethylethanolamine of dog lung tissue and the lecithin associated with pulmonary surfactant lipoprotein had similar fatty acid patterns suggesting a product : precursor relationship. GLUCK AND SRIBNEY~ using homogenates of lung from rabbit fetuses found that incorporation of radioactive choline into lecithin declined and that the methyl group of [Me-W]S-adenosylmethionine increased shortly prior to term. The conclusion drawn from these data by others” has been that perhaps the major pathway of surfactant lecithin synthesis occurs via methylation of phosphatidylethanolamine in the granular pneumoncytes. The present study was undertaken to elucidate some of the aspects of the biosynthesis in vizroof lung lecithin. Biockim. Biopk~~s. Acta, 152 (1968) 552-558

LUNG LECITHIN SYNTHESIS in. ViWJ

553

MATERIAL AND METHODS Female Sprague-Dawley rats (weighing 200-250 g) maintained on standard lab chow were used in all experiments. From the time of isotope administration until sacrifice by cervical dislocation the animals were allowed free access to food and water. At time of sacrifice lungs and liver were quickly removed, washed in cold 0.99;; NaCl, homogenized, lipids extracted and the phospholipids characterized by methods reported elsewhere 6t6. Briefly, after separation of phospholipids by silicic acid column chromatography, all compounds were checked for purity by the thin-layer chromatographic

method

of SKIPSKI et al.? using IO O/ 1’0 H,SO,

detection of organic material. [Jle-3H]Choline (IOO mC/mmole)

spray followed by charring

and L-[hfeJ4C]methionine

for

(10.4 mC/mmole)

were purchased from New England Nuclear Corp. [r,s-‘%,]Choline (20 mC/mmole) was purchased from Schwarz BioResearch, Inc. Appropriate mixtures of radioactive material

were made up in sterile 0.97~ NaCl, and I ml injected

attempt

was made to determine

the amount

of activity

ilia the tail vein. No

remaining

in the syringe

or

lost by bleeding. Radioactivity was determined in a Nuclear-Chicago Mk. I liquid scintillation spectrometer as described in detail by BALINT et al.@. Fatty acids were determined

using an F & M Model 402 gas chromatograph

describeda.

All reagents

were of analytical

by methods

previously

grade.

RESULTS AND DISCUSSION In separation the

of lung phosphatides

phosphatidylethanolamine

chromatography

peak

of this fraction

spot corresponded

to standard

significant

eluted

revealed

radioactivity

off a silicic

acid

was detected

column.

two spots with iodine vapor.

phosphatidylethanolamine

in

Thin-layer

and contained

The larger only trace

radioactivity. A smaller second spot ran just behind the phosphatidylethanolamine, was ninhydrin negative and contained 957, of the radioactivity detected in the phosphatidylethanolamine

peak eluted off the silicic acid column.

FIALKOW’ have identified Radioactivity

this material

was also detected

in the phosphatidylethanolamine

eluted off a silicic acid column. Thin-layer spot, phosphatidylethanolamine, having thin-layer

plate just behind

MORGAN, FINLEY AND

as phosphatidyl-N,N-dimethylethanolamine. fraction

from liver

chromatography however revealed a single a trace of radioactivity. The area on the

phosphatidylethanolamine

and corresponding

to phos-

phatidyl-N,N-dimethylethanolamine contained 955(, of the radioactivity detected in the phosphatidylethanolamine peak eluted off the silicic acid column. Table I gives the distribution of L-(Me-lK]methionine and [ICfe-3H]choline incorporation between the choline phosphatides and phosphatidyl-N,N-dimethylethanolamine in lung and liver. The amount of [MQH]choline activity in phosphatidyl-N,N-dimethylethanolamine was unexpected. To determine whether the radioactivity detected in this spot might be due to an unusual lecithin species eluted from the silicic acid column along with phosphatidyl-N,N-dimethylethanolamine, double label choline studies were performed using [r,z-14C,]choline and [Me-3H]choline (see Table II). [Me-3H]Choline incorporation into phosphatidyl-N,N-dimethylethanolamine was similar to that in Table I. [r,z-14C]Choline was detected at twice background (14C background 16 counts/min) and was not considered significant. RioChim.

Bioph_Vs.

.4Cfa,

152

(196s)

552-558

H. L. SPITZER, K. MORRISON, J. R. NORMAN

554 The finding of [MGH]choline amine could be expected according GREENBERG'~ direct

activity in phosphatidyl-N,N-dimethylethanolto the scheme proposed by BREMER, FIGARD AND

in which the loss of the first methyl

transmethylation

between

betaine

group in choline

and homocysteine

catabolism

to form

is a

methionine.

Similar data regarding choline-methyl group reutilization in methylation of phosphatidylethanolamine have been reported by GROTH, BAIN AND PFEIFFER". Betaine TABLE

1

DISTRIBUTIONOF L-[M&%]METHIONINE LIVER

AND [Me-~H]CHOLINE RADIOACTIVITY IN

LUNG

AND

PHOSPHATIDES

Values are expressed in disint./min applied to a silicic acid column. Phosphatide Phosphatidyl-N,Ndimethylethanolamine* Choline Choline phosphatides**

of isolated

material from I mg of phospholipid

phosphorus

Liver

Time (h)

LLl?‘lg Choline

Methionine

Ckoline

Methionine __-

2 4 2 4

5445 3935 4099280 4266542

851 1050 18080 27802

14974 25078 1398348 1573830

424” 3 445 542 180 534672

* Phosphatidyl-N,N-dimethylethanolamine is a tentative identification based on comparison of our thin-fayer chromatographic data with that obtained by MORGAN, FINLEY AND FIALKOW~. This phosphohpid has been designated Px by other investigators.*,9 MORGAN, FINLEY AND FIALKOW~ identified Px as phosphatidyl-N,N-dimethylethanolamine by gas-chromatographic analysis of the base. ** Choline phosphatides: lecithin, lvsolecithin and sphingomyelin. Female rats were given simultaneously an intravenous injection-of 25 PC L-[Me-Ylmethionine and IOO ,uC [IUe-3Hjcholine. Results are from pooled tissue from 3 animals.

has been shown to be as effective of phosphatidylethanolamine12. (Table

a methyl

donor as methionine

The inability

to detect

II) in phosphatidyl-N,N-dimethylethanolamine

MUNTZ~~that phosphatidylcholine ethanolamine. Lecithin (a) methylation

synthesis

in mammalian

of phosphatidyl

confirms

is not catabolized

in the methylation

[~,a-lJC,]choline

radioactivity

the original work of

to phosphatidyl-N,N-dimethyl-

species takes place via two major pathways*;

ethanolamine

with methionine

being the donor of

of choline via cytidine diphosphate all three methyl groups10y15 or (b) incorporation compare choline (CDP-choline) 16. The present study was designed to simultaneously these pathways in lung and liver using L-[A1G4C]methionine and [I11e3H]choline. By calculating the ratio of [AlGH]choline to L-[,lle-l”C]methionine incorporation into lecithin one is able to approximate the relative activity of these two pathways in one tissue and compare it to another tissue. Table III gives the specific activity (disint./ min per patom lipid P) of L- [dle-l%]methionine and [&Is-3Hlcholine incorporation into lung and liver lecithin. The ratio of [AWe-3Hlcholine to L-[Ale-Klmethionine incorporation into lecithin isolated from liver is 2.5 at z h and 2.9 at 4 h, while in lung the ratios are 250 and 160. The values obtained for liver are in good agreement to Lwith those reported in the literature6917. Since the ratio of [A$e-JH]choline [Ale-lJC]methionine is IOO times greater in lung than in liver lecithin it would appear * A third pathway has been described I4 direct incorporation of free choline into lecithin. This pathway would give identical results as those assigned to the CDP-choline pathway in the present study. Biochiw.

BzopJr~~s. .Icta,

152 (1968) 552-558

LUNG

LECITHIN

TABLE

SYNTHESIS

i!ZViVO

555

II

DISTRIBUTION Values

are

applied

to

OF [&I&3H]CHOLINE expressed a silicic _

in acid

AND

disint./min

of

[I,~-*4C,]~~~~~~~ isolated

Animal

Phosphatidylethanolamine

3H ‘4C

Phosphatidyl-N,NCholine phosphatides** See

Table

***

Radioactivity

rats

were

and

12.5 &

that

given

IN LUNG

PHOSPHATIDES

phospholipid

phosphorus

***

3H

7538987

*** II

150532

3

I423 5838

13172

1%

A4xiwal

3083

71’54

***

16.355

274248

9889591

23S711

199493

definition.

was

detected

simultaneously

[I,z-K,]choline

methylation

pathway

of

***

II71

14C

I for

I mg

A4i2imzl 2

1

7954

3H

dimethylethanolamine*

*. **

from

column.

Phosfihatide

Choline

RADIOACTIVITY

material

at twice

background

an intravenous (20

injection

was

of IOO$

not

considered

significant.

[Me+H]choline

(IOO

Female

mC/mmole)

mC/mmole).

of phosphatidylethanolamine

of lecithin

and

synthesis

may not be the major

biosynthetic

in lung.

There are several possible explanations

for the differences

observed

in lecithin

synthesis in lung and liver: (a) Lung tissue receives lecithin intact from the liver via plasma. (b) There is a selective dilution or uptake of choline or methionine by lung or liver tissue. (c) The major pathway of lecithin synthesis in lung tissue is via the CDPcholine pathway and not via methylztion of phosphatidylethanolamine. (d) Lung lecithin

is a mixture

of molecular

over of lung tissue methionine principle in lung.

donor of the methyl

speci 2s synthesized via different pathways. (e) Turnmethyl is relatively slow. (f) Methionine is not the

group in the methylation

of phosphatidylethanolamine

In a study of lecithin synthesis using similar methodsBy17, it was found that equilibration of liver and plasma lecithin takes place at about 2 h. If the choline of lung tissue lecithin is derived from plasma lecithin the [Me-3H]choline to L- [Ale-Xlmethionine ratio should be ,similar to that found in liver. Since the ratio is always greater

than IOO in lung over the time period studies and less than 3 in the liver it is

difficult to believe that there is a relationship data tend to exclude the first possibility from the liver via plasma.

between

lung and liver lecithin.

(a) that lung tissue receives

There is 3 times more [,WGH]choline phatides (Table I) of lung than liver. Specific

lecithin

These intact

incorporation into the choline phosactivity (Table III) is greater in lung

lecithin than in liver lecithin. BJDRNSTAD AND BREMER'? using intraperitoneal injection of [I,z-14Cz]choline found incorporation into choline phosphatides greater in liver than lung. This difference is thought to be due to the mode of tracer administration. In parallel experiments the same investigators gave paired animals [I,z-14C,]choline plus unlabeled

choline.

Liver

and lung incorporation

was reduced

7596,

suggesting

similar dilution in both tissues. At present, there is no detailed evidence regarding selective dilution or uptake of choline or methionine by either lung or liver. This possibility (b) cannot be excluded. The incorporation of L- [Me-Xlmethionine into choline phosphatides was 20 times greater in liver than lung (Table I). Unlike choline incorporation, this difference Liver microsomes incubated with is present in animals injected intraperitoneally17. Riochinz.

Biophvs.

.-lcta,

152

(1968)

552-558

H. L. SPITZER, K. MORRISON, J. R. NORMAN

556 TABLE

III

SPECIFICACTIVITIESOF LUNGAPZD ~IETHIONINE Specific in

AND

activity

Table

LIVER

LECITHIN

.4FTER

INTRAVENOUS

INJECTION

OF L-[fife-‘%-

~M~~HJCHOLINE =

diSint./min

per

@om

lipid

P.

Experimental

conditions

are

the

Same

as stated

I.

Lung Cholirze

Time (11) 2

207 151

4

240218

Methioniv~r 828

-

I498

(,41e-14C]S-adenosylmethionine choline

phosphatides

LkW Choline

Methionirle

69487 82 520

2773.3 28319

incorporate

methylation of phosphatidylethanolamine reported that the ability of lung tissue ethanolamine

65 times

than lung microsomesls.

increases

markedly

more

methionine

methyl

into

Such data in vitro tend to show that

is greater in liver than lung. It has been homogenates to methylate phosphatidyl-

during gestation

with a concomitant

decline in the

activity of the CDP-choline pathway3. Since no data on net synthesis was given in this study one cannot determine the contribution of each pathway to lecithin synthesis. These data support the possibility (c) that the major pathway of lecithin synthesis ipt Go in lung tissue is via the CDP-choline pathway and not via methylation of phosphatidylethanolamine. The lecithin fatty

of lung tissue is composed

acids esterified

are palmitate

of a variety

to the glpcerylphosphorylcholine

43:/a, stearate

189;,

palmitoleate

of molecular

backbone

6y6, oleate

species.

The

in rat lung lecithin

1776, linoleate

694 and

arachidonate II:,;. These values are very similar to those reported by MORGAN, FINLEY AND FIALKOW’ for the lecithin of dog lung tissue. 75;$ of the fatty acids esterified to the surfactant lecithin are saturatedly19 indicating that it is probably a single molecular [Il1GH]choline tionated

species. Fig. I shows the pattern of phospholipid and L-I,Ue-“Clmethionine incorporation into

on a silicic acid column.

The fact that

the specific

phosphorus elution, lung lecithin frac-

activities

of LAVe+H]-

disint./min per @ml P

Fig. I. Silicic acid column elution of 4 h lung lecithin from Table I. 10 ml fractions were collected; 0.5 ml was taken from every other fraction for phosphorus determination. At the points indicated 5.ml aliquots were dried down and radioactivity determined. Phosphorus (O----O); specific activities (disint./min per ,ugatom lipid P) of [MeQH]choline (x---x ), L-[MG4C]methionine (A-A). (Sphingomyelin, [MGH]choline roe I 73 disint./min per /“atom lipid P, L- [Me-W]methionine 1008 disint./min per ,uatom lipid P.)

Biochim. Biophvs. .dcta. I j2 (1968)

552-558

LUNG LECITHIN

SYNTHESIS

in ViVO

choline

and L-[Me-14C]methionine

elution

argues very strongly

557 do not parallel

that

lung lecithin

each

other

nor the phosphorus

is a heterogeneous

mixture.

Similar

of liver lecithin. Also, have been reported by othersZ0q2r in fractionation studie+ of lecithin elution from a silicic acid column have shown that containing the most unsaturated fatty acids are eluted first and lecithins

findings detailed lecithins containing

mostly

saturated

fatty

acids are eluted

last.

Thus

it appears

that

the

lecithin of lung tissue is a heterogeneous mixture (possibility (d) above) and the highly unsaturated lecithins are synthesized viu methylation of phosphatidylethanolamine while the highly

saturated

No final conclusions,

lecithins

however,

are synthesized

concerning

via the CDP-choline

the lecithin of surfactant

pathway.

can be made until

it and the appropriate molecular species in lung tissue are compared. The finding of L-[Me-14C]methionine radioactivity in phosphatidyl-N,N-dimethylethanolamine

isolated

from

lung suggests

that

this compound

mediate in the stepwise methylation of phosphatidylethanolamine. L-[Me-laC]nlethionine incorporation (Table I) into this compound gests that the turnover these time periods mediate.

of methionine

one would expect

BALDESSARINI~~ has assayed

onine. Liver contains

methyl

is an inter-

The increase in from z to 4 h sug-

in the lung may be relatively

to see maximum a number

incorporation

slow. At

into an inter-

of rat tissues for S-adenosylmethi-

26 pg/g wet weight while lung contains

II pug/gwet weight. This

would tend to exclude the lack of immediate precursor for slow methionine methyl turnover but might imply less methyltransferase enzyme in lung tissue (possibility (e) above). Using tidylethanolamine of ethanolamine to exclude

[r,z-r4C,]ethanolamine there is much less conversion of phosphato lecithin in lung as compared to liver”. The similar incorporation and the methyl

the possibility

in methylation

group of methionine

of a methyl

of phosphatidylethanolamine

Lung lecithin

is a heterogeneous

into lung lecithin”

donor other than methionine in lung (possibility

mixture

of molecular

would tend

being involved

(f) above).

species synthesized

by

both the methylation of phosphatidylethanolamine and the CDP-choline pathway. It appears under the conditions of the present experiment ilz z&lo that methylation of phosphatidylethanolamine lecithin. synthesis

The data further

is of minor importance

in the synthesis

of lung tissue

show that liver is not the source of lung lecithin

occurs in situ mainly

Z&Zthe CDP-choline

and that

pathway.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the encouragement and guidance of Dr. J. M. MCKIBBIN throughout this investigation as well as the editorial criticisms of Dr. G. SACHS. This study was supported by a grant from the National Science Foundation GB-4765, U.S. Public Health Service Training Grant in Gastroenterology ~A-5286 and U.S. Public Health Service General Support Grant FR 05349. A portion of this work was conducted in the Clinical Research Center Laboratory of the University of Alabama Medical Center supported by the National Institutes of Health Grant ZMOI-FR-3207.

Biochim.

Riopl~ys.

Acta,

151 (1968)

552-558

H. I.. SPITZER,

55s

I<. MORRISON,

J. R. NORMAN

REFERENCES I z 3 4 5 6 7 8 g IO II IZ 13

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Biochim.

Bioph_v~. .4cfa, 152 (1968) 552-558