Lipid Synthesis by Perfused Lung ELIAS G. TOMBROPOULOS 1 and JOHN G. HADLEY, Biology Department, Battelle, Pacific Northwest Laboratories, Richland, Washington 99352 ABSTRACT

An isolated lung ventilated with pulses of negative pressure and perfused through the pulmonary vasculature was utilized for the study of 3-sn-phosphatidylcholine synthesis. The perfusion fluid consisted of a Krebs-Ringer phosphate buffer with 6% bovine serum albumin, pH 7.4, and the appropriate substrate. The simultaneous incorporation of (1-14C) palmitate and (2-3H) glycerol and the simult a n e o u s incorporation of (CH3-14C) choline and (CH3-3H) methionine were examined. F r o m these experiments it is concluded: 1) lung tissue incorporates (2-3H) g l y c e r o l into 3-sn-phosphatidylcholine to a greater extent than any other lipid examined; 2) both choline and methionine contribute to the synthesis of 3-sn-phosphatidylcholine, and 50-70% of the label in its nitrogen base is derived from choline and 30-50% from methionine; and 3) a high PO 2 appears to reduce the synthesis of 3-sn-phosphatidylcholine. INTRODUCTION

The prevention of alveolar collapse during expiration is primarily due to the presence of a surface active substance known as lung or p u l m o n a r y surfactant (1,2). Under certain pathological conditions, inadequate surfactant or abnormal surfactant appears to occur. To understand these pathologies, it may be necessary to examine the normal lung tissue synthesis of 3-sn-phosphatidylcholine (PC) and to identify the regulatory mechanisms involved. Most of the surface active properties of lung surfactant are due to the saturated phospholipid component of the material, dipalmitoyl3-sn-phosphatidylcholine (d~palmitoyl-PC) (3). There are contradicting reports in the literature as to whether lung tissue synthesizes this material primarily do novo, or whether it modifies (as to fatty acid composition) the PC supplied by the blood (4-7). Blood PC containing at least one unsaturated fatty acid might be modified in the lung by a combination of a deaeylation-acylation cycle and lung lipase (8). It has also been suggested that blood 3-sn-lysoIpresent address: Division of Nutrition, HFF-268, Bureau of Foods, Food and Drug Administration, 200 "C" Street, S.W., Washington, DC 20204.

phosphatidyl-choline (lyso-PC) may be converted to PC by the lung (7). Lung tissue has the ability to synthesize PC de novo, but disagreement exists as to the relative contributions of the two main pathways (i.e., methylation vs. cytidine monophosphate-phosphorylcholine) to the formation of the nitrogenous base in the de novo synthesized PC (9-12). The synthesis of PC has been studied in subcellular particles, lung tissue slices, and whole animals. It is possible that these previous studies concerning themselves with systems that no longer are integrated (subcellular particles and tissue slices) or with systmes that cannot be easily controlled (whole animals) do not reflect the PC synthesizing ability of the lung as an organ. In this communication, an isolated, perfused lung preparation has been employed to examine t h e i n c o r p o r a t i o n of glycerol, palmitate, choline, and the methyl group of methionine into PC. Similar preparations have been utilized r ently by others for biochemical studies (13). It is concluded from the present experiments: 1) lung tissue has a high capacity to synthesize phosphatidylcholine de novo, 2) ca. 1/3 of the nitrogenous bases of the newly synthesized PC are formed via the methylation pathway, and 3) enriched oxygen atmospheres tend to inhibit the de novo synthesis of the PC at the level of nitrogenous base formation. MATERIALS AND METHODS Animals

Two- to three-month old female Wistar rats and Syrian golden hamsters were used. The Wistar rats came f r o m Hilltop Laboratory Animals (Scottsdale, PA). Hamsters were from Engle l a b o r a t o r y Animals, Inc. (Farmersburg, IN). Animals were isolated for acclimation and for detection of incipient infections at least 3 weeks before being used. They were fed water ad libitum and Wayne Lab-Blox from Allied Mills, Inc. (Chicago, 1I,). The Lab-Blox contains 24.5% protein, 4.15% fat, and 49.7% nitrogenfree extract. Materials

Choline chlOride (4.99 mCi/mmol) (1,214C) a n d glycerol ( 5 0 0 m C i / m m o l ) (2-3H) were purchased from New England Nuclear (Boston,

491

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E.G. TOMBROPOULOS AND J.G. HADLEY

FIG. 1. Schematic diagram of perfused lung preparation. MA); methionine (1,000 mCi/mmol) (L-Me-3 H) from International Chemical and Nuclear Co. (Irvine, CA); and palmitate (45.8 mCi/mmol) (1-14C) from Volk Radiochemical Co. (Burbank, CA). Unlabeled choline chloride was purchased from Sigma Chemical Co. (St. Louis, MO); L-methionine from Nutritional Biochemical Co. (Cleveland, OH); glycerol from Mallinckrodt Chemical Co. (St. Louis, MO); and palmitic acid from Fisher Scientific Co. (Fairlawn, NJ). The unlabeled materials were used for maintaining approximately optimal concentrations of the substrates in the study. B o v i n e serum albumin fraction V was obtained from Miles Lab., Inc. (Kankakee, IL); silica gel without binder for the thin layer chromatography (TLC) of phospholipids from Camag (Muttenz, Switzerland); and Silica Gel G from TLC of neutral lipids from E. Merck, A.G. (Darmstadt, West Germany). Neutral lipid standards used for TLC were purchased from The Hormel Institute (Austin, MN) and phospholipid standards from Applied Science Laboratories, Inc. (State College, PA). The Aquasol scintillation cocktail used for radioassay was purchased from New England Nuclear Pilot Chemical Division (Boston, MA). Lung Perfusion

The basic lung perfusion preparation and methods of Rosenbloom and Bass (14) were used. The preparation consisted of the excised lung and trachea of a hamster or rat, ventilated with pulses of negative pressure, while being perfused through the pulmonary vasculature. A simplified schematic drawing of the preparation is shown in Figure 1. The lung was housed in a glass container and suspended from the plexiglass lid through which pass the cannulae for the trachea and pulmonary artery. Pulses o f negative pressure were exerted in the chamber, LIPIDS, VOL. 11, NO. 7

causing the lung to inflate. Deflation was allowed by bleeding air back into the chamber between inflations. The trachea was attached to a humidified supply (at ambient pressure) of either room air or oxygen-enriched air. The perfusion fluid was drawn from a reservoir beneath the lung by a peristaltic pump and passed to the top of the H-shaped column. The fluid filled the column to the horizontal bar of the H. The height of this bar maintained the constant perfusion pressure, while media in excess of that flowing through the lungs returned to the reservoir via the other leg of the H column. The perfusion fluid entered the lung through a cannuta tied into the pulmonary artery. The fluid was oxygenated through the lung as occurs physiologically. After coursing throughout the lung vasculature, it left the lung via the pulmonary veins and dropped to the reservoir. The entire system was enclosed in a temperature-controlled box. The basic perfusion fluid consisted of a Krebs-Ringer phosphate or bicarbonate buffered solution containing 6% bovine serum albumin. Two potential energy and carbon sources were used in the course of these studies: glucose (0.05 M) in the case of nitrogenous base incorporation, and citrate (0.05 M) in the case of glyceride and palmitate. Rates of either bases or palmitate and glycerol incorporation into glycerides were approximately the same. This would imply that either (a) both materials serve as carbon source, or (b) endogenous levels of carbon and energy sources in the cells are such that added glucose or citrate did not affect energy or carbon requiring processes. I n the experiments described here, the preparations were maintained at 30 + 2 C, and the perfusion pressure was a constant 15 cm H20. The tidal volume was 1-1.5 cc for hamsters and 2-2.5 cc for rats, and the respiration rates were 70-+ 5/min for hamster lungs and 60 + 5/min for rat lungs. The perfusion flow r a t e s w e r e 4 . 0 - 4 . 5 ml/min for rats and 2.0-2.5 ml for hamsters. The volume of perfusate was 15 ml for each lung. The concentration of the substrates in the perfusion fluid were 1.4 mM palmitate, 5 mM glycerol, and 2 mM nitrogenase base or methyl donors. In preliminary experiments in our laboratory, representative sections o f lung tissue were taken from perfusion preparations after either 10, 20, 30, 45, 60, or 90 rain of perfusion. Sections were placed in 10% neutral buffered f o r m a l i n a n d subsequently processed by standard procedures and stained with hematoxylin and eosin. Light microscopic examination failed to reveal any abnormal tissue and ceil structure. As an indication of metabolic

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493

TABLE I Simultaneous Incorporation of (1-14C) Palmitate and (2-3H) Glycerol into Lipids by Perfused Hamster Lunga Perfusion period (min)

Number of experiments Labled substrate

10 10

3 3

10

3

40 40

5 5

40

5

(2-3H) Glycerol (1-14C) Palmitate (2-3H) Glycerol a (1-14C) Palmitate (2-3H) Glycerol (1-14C) Palmitate (2-3H) Glycerol c (i.14)pa_!mitate

1,2-DG

nmol/500 mg lung tissue b TG PC

2.3 + 1.7 9.2 • 3.9

2.7 --. 1.3 5.5 • 1.9

0.2 • 0.1

0.2 -+ 0.1

3.5 • 1.2 10.7 • 1.7

6.5 -+ 2.0 40.6 • 7.8

0.3 • 0.1

0.2 +- 0.5

21.8 -+ 4.2 20.3 + 6.8 0.8 -+ 0.05 76.5 • 2.7 107.0 + 8.4 0.7 +- 0.03

PE 5.8 -+ 0.9 5.8 • 0.8 1.2 + 0.2 17.2 • 2.1 18.7 • 1.8 1.0 +- 0.1

aperfusion conditions are described in MateriaLs and Methods. Phosphate buffer was used as the perfusion fluid containing 6% bovine serum albumin, 0.05 M citrate, 1.4 mM palmitate, and 5 mM glycerol. The flow rate was 2.0-2.5 ml/rnin and the total perfusate volume 15 rnl. Results are expressed as means _+SEM between experiments. b1,2-DG = 1,2-diacyl-sn-glycerol, TG = triacyl-sn-glycerol, PC = 3-sn-phosphatidylcholine, PE = 3-sn-phosphatidylethanolamine. CThe ratio was calculated from the individual observations. integrity, t h e l i n e a r i t y o f a c e t a t e i n c o r p o r a t i o n i n t o f a t t y acids a n d t h e lactic acid p r o d u c t i o n from glucose b y t h e p e r f u s e d lung were e x a m i n e d . Weights o f t h e l u n g p r e p a r a t i o n s were t a k e n b e f o r e a n d a f t e r p e r f u s i o n as an i n d i c a t i o n o f e d e m a . D u r i n g t h e actual experim e n t s , t h e i n t e g r i t y of t h e lung was j u d g e d b y its e x t e r n a l a p p e a r a n c e , ease a n d m o d a l i t y o f r e s p i r a t i o n , a n d changes in weight. Results are r e p o r t e d o n l y f r o m t h o s e lungs w h i c h a p p e a r e d normal during perfusion in all criteria examined.

Analytical Procedures At t h e e n d of t h e e x p e r i m e n t a l p e r f u s i o n period, t h e lungs were p e r f u s e d w i t h saline to r e m o v e t h e labeled p e r f u s i o n fluid f r o m t h e large vessels. T h e lungs were t h e n s t r i p p e d t o r e m o v e t h e t r a c h e a a n d large b r o n c h i a n d h o m o g e n i z e d in c h l o r o f o r m : m e t h a n o l ( 2 : 1 ) . E x t r a c t i o n o f lipids, t h e i r f r a c t i o n a t i o n b y TLC, a n d radioassay o f s a m p l e s were p e r f o r m e d as previously described (15). S i m u l t a n e o u s m e a s u r e m e n t of 3H a n d I 4 C in t h e d o u b l e - l a b e l e x p e r i m e n t s was m a d e in a t w o - c h a n n e l l i q u i d scintillation c o u n t e r (5). The f a t t y acid c o m p o s i t i o n o f t h e individual lipid classes was d e t e r m i n e d f r o m t h e corr e s p o n d i n g b a n d s of t h e T L C plates. By u s i n g long ( 4 0 c m ) TLC plates, we a v o i d e d cont a m i n a t i o n of t h e e x a m i n e d lipid classes w i t h a n y d e t e c t a b l e a m o u n t o f e t h e r lipids. Silica gel b a n d s c o n t a i n i n g t h e lipids were scraped i n t o s c r e w - c a p p e d test t u b e s c o n t a i n i n g 4% H 2 S O 4 i n a n h y d r o u s m e t h a n o l a n d were left o v e r n i g h t

a t r o o m t e m p e r a t u r e . Water, f o l l o w e d b y p e t r o l e u m e t h e r , was t h e n a d d e d t o t h e tubes. T h e u p p e r p h a s e c o n t a i n i n g t h e esters was t h e n evaporated to dryness under a stream of nitrogen. T h e esters were a n a l y z e d using a gas chromatograph e q u i p p e d w i t h dual f l a m e i o n i z a t i o n detectors. T h e glass c o l u m n , 1.83 m b y 2 m m inside d i a m e t e r , p a c k e d w i t h 15% d i e t h y l e n e glycol s u c c i n a t e o n 100-120 m e s h Gas C h r o m P, was o p e r a t e d at 190 C w i t h a n i n l e t pressure o f 1.1 k g / c m 2. Q u a n t i t a t i v e data were c a l c u l a t e d b y t h e f o r m u l a Rt x h, w h e r e R t is t h e r e t e n t i o n t i m e a n d h is t h e p e a k h e i g h t (16).

RESULTS For clarity, t h e results have b e e n divided i n t o t w o m a i n parts. T h e first part i n c l u d e s results o n t h e m o d e of f o r m a t i o n o f t h e 1,2diacyl-sn-glycerol ( 1 , 2 - D G ) m o i e t y o f lipids a n d the second with the mode of formation of the n i t r o g e n o u s base o f PC a n d t h e effect o f high PO 2 o n its f o r m a t i o n .

Simultaneous Incorporation of (2-3H) Glycerol and (1-14C) Palmitate If the incorporation of (2-3H) glycerol is indicative of de n o v o s y n t h e s i s o f glycerides, t h e n t h e l u n g tissue s y n t h e s i z e s over 5 t i m e s m o r e p h o s p h o l i p i d s t h a n n e u t r a l iipids ( T a b l e I). This d i f f e r e n c e b e c o m e s greater w i t h t i m e b e c a u s e t h e rate of s y n t h e s i s o f n e u t r a l lipids decreases a f t e r 10 rnin a l t h o u g h t h a t of p h o s p h o l i p i d s r e m a i n s a b o u t t h e same. Saponi-

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494

TABLE II Fatty

Acid Composition of Major Lipid Classes from Perfused Hamster Lungsa PC

14:0 16:(B) c 16:0 16:1 18:0 18:1 18:2 18:3 20:4 22:6

% of Total fatty acidsb PE

1.5 + 0.02 0.4 + 0.02 53.2 + 1.2 4.7 + 0.2 10.0 + 0.0 14.1 • 0.7 6.7 + 0.4 4.5 • 0.5 4.8 • OA

1.7 6.5 23.4 3.8 23.9

+ 0.5 • 2.0 + 1.8 + 0.8 + 1.1

26.1 • 0.9

11.5 • 1.2 tr

tr ?

TG 1A + 0.03 0.3 • 0.02 25.8 + 1.1 6.4 + 0.4 5.0 • 0.5 34.8 + 0.7 • 0.7 tr

24.1

2.2 • 0.1

a F a t t y acid composition of t h e three major lipid classes isolated from hamster lung in which the simultaneous incorporations of (2-3H) glycerol and (1-14C) p a l m i t a t e w e r e examined. The results are expressed as mean percentage • SEM of three experiments. bpc = 3-sn-phosphatidylcholine, PE = 3-sn-phosphatidylethanolamine, TG = triaeyl-snglycerol.

Cprobably branched chain not definitely identified. fication of lipids and isolation of fatty acids neutral lipids. indicated that no more than 5-8% of the 3H The fatty acid analysis (Table II) indicates activity was incorporated into the fatty acid that PC and PE are not derived from the same fraction. The highest incorporation of glycerol PA pool because they do not have the same occurred in PC, a result which would appear fatty acid composition. The incorporation of contrary to that observed with subcellular frac- glycerol paralleled that of palmitate into PC, tions (5). The (2-3H) glycerol: (1-14C) palmi- resulting in an almost constant (2-3H) glycerol: tare incorporation ratio serves as an indication (1-14C) palmitate ratio. as to whether palmitate incorporation is occurring via synthetic versus exchange reactions and Formation of the Nitrogenous Base of PC It was observed that in the experiments whether the various lipid fractions are derived utilizing the bicarbonate buffer, a rapid increase from a common 3-sn-phosphatidic acid pool. In separate experiments not reported here, in the pH of the perfusate occurred unless acid the ratio of glycerol to palmitate incorporation was periodically added to the perfusate reserwas the same between 1,2-DG and 3-sn-phos- voir. The loss of CO2, both into the alveolar phatidic acid (PA). Therefore, if the labeling of spaces and from the open end of the constant the isolated 1,2-DG represents the pattern of pressure column, invariably led to a decreasing [H +] of the perfusate. For experimental conlabeling of a c o m m o n PA pool from which both the neutral and phospholipids are derived, then venience, a phosphate buffer was employed for the (2-3H) glycerol: (1-14C) palmitate ratio the following experiments, eliminating the need should be the same in 1,2-DG and phospho- for titration of the perfusate to maintain lipids. The triglyceride (TG), having an addi- physiological pH. In addition, use of phosphate tional fatty acid (three instead of the two for buffer not saturated with a gaseous atmosphere 1,2-DG and phospholipids), should have a allowed us to examine the effects of breathing (2-3H) glycerol: (1-14C) palmitate ratio 1/3 enriched 02 on PC synthesis, uncomplicated by smaller than that of 1,2-DG (0.2vs. 0.3), as problems associated with other dissolved gases. would be expected if they were derived from a Nevertheless, because bicarbonate buffer is common 3-sn-phosphatidic acid and fatty acid most commonly used for studying lipid synpool. The ratio of 3H:14C at 40 min is used thesis, some experiments were done with because these numbers represent values ob- bicarbonate for comparison (Tables III and IV). tained after equilibration o f the perfusion The results using bicarbonate (Table III) and media and the lung has occurred. Shorter per- phosphate buffer (Table IV) appear compatible fusion periods may not permit complete equili- to the incorporation of the nitrogenous base bration. Both PC and 3-sn~ and their ratios. amine (PE), on the other hand, have a much From the data in Table III, the relative conhigher (0.7 and 1.00) 3H:14 C ratio and thus do tribution of the two pathways may be estinot appear to utilize the same pool as the mated by following the simultaneous incorporaLIPIDS, VOL. 11, NO. 7

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495

TABLE III Simultaneous Incorporation of (CH3-3H) Methionine and (CH3-14C) Choline into 3-sn-Phosphatidylcholine by Perfused Hamster Lung a nmol/0.5 g lung Perfusion period (rain) 10 30 60 90

(CH3-3H) Methionine

(CH3-14C) Choline

4.5 18.4 33.1 59.0

6.0_+ 1.9 22.7 • 7.7 37.0 • 6.5 78.9 • 14.5

• 0.2 • 3.4 • 2.6 • 10.6

(CH3"3H) Methi~ CH 3-14C) Choline 0.3 0.9 0.7 0.8

• • • •

0.2 0.2 0.1 0.1

aperfusion conditions are described in Materials and Methods. Bicarbonate buffer was used in the perfusion fluid containing 6% bovine serum albumin, 0.05 M glucose, and 2 mM nitrogenous base. The flow rate was 2.0-2.5 ml/min and the total perfusate volume 15 ml. Results are expressed as means of four experiments + SEM. bThe ratio was calculated from the individual observations. TABLE IV Effect of the Composition of Inspired Air on Incorporation of (CH3-14C) Choline and (CH3-3H) Methionine into 3-sn-Phosphatidylcholine by the Pefrused Lung of Rats and Hamsters a n m o l / g rat lung or 0.5 g o f hamster lung

Animal species Hamster Rat

Composition of inspired air Room air 9 5 % 0 2 + 5%CO2 c Room air 9 5 % 0 2 + 5%CO 2

(CH3-3H) Methionine

(CH3-t4C) Choline

20.1 15.9 58.7 38.1

38.2 25.5 116.9 71.0

-+ 2.4 -+ 0.6 + 2.8 • 2.5

(CH3"3H) Methi~ (CH3 -14C) Choline

-+ 4.7 -+ 1.8 + 6.9 + 7.1

0.5 0.6 0.5 0.6

+ 0.02 + 0.02 -+ 0.01 -+ 0.04

aperfusion conditions are described in Materials and Methods. Perfusion period was 40 min. Phosphate buffer was used in the perfusion medium containing 6% bovine serum albumin, 0.05 M glucose, and 2 mM nitrogenous base. The flow rate for the rats was 4.0-4.5 ml/min, for the hamsters 2.0-2.5 ml/min, and the total perfusate base. Results are expressed as mean of three experiments + SEM. bThe ratio was calculated from the individual observations. COnly two experiments were conducted. tion of choline and the methyl group of m e t h i o n i n e i n t o PC. T h e m e t h y l a t i o n p a t h w a y w o u l d a p p e a r t o be of great i m p o r t a n c e , as it was r e s p o n s i b l e f o r f r o m 4 0 % t o 50% of t h e t o t a l de h o v e s y n t h e s i s of PC w h e n b i c a r b o n a t e was u s e d as a b u f f e r ( T a b l e III) a n d ca. 35% w h e n p h o s p h a t e was e m p l o y e d . T h e ( C H 3 - 3 H ) methionine:(CH2-aH) choline ratio after 10 rnin was n e a r l y c o n s t a n t f o r all t h e p e r f u s i o n p e r i o d s s t u d i e d ( T a b l e III). E x p o s u r e o f c e r t a i n a n i m a l species t o increased p a r t i a l pressures o f 0 2 leads t o a v a r i e t y of p a t h o l o g i c a l , histological, a n d b i o c h e m i c a l a l t e r a t i o n s in t h e lung, i n c l u d i n g d e c r e a s e d d i p a l m i t o y l - P C c o n c e n t r a t i o n s (17). It was therefore considered important to examine the effect o f o x y g e n c o m p o s i t i o n o f r e s p i r e d air o n t h e p e r f u s e d lung. A f t e r 4 0 m i n o f p e r f u s i o n , t h e lungs respiring t h e O 2 - e n r i c h e d a t m o s p h e r e s showed a reduction of synthesis by both pathways ( c o m p a r e d t o t h o s e r e s p i r i n g r o o m air) ( T a b l e IV). A l t h o u g h t h e n u m b e r o f a n i m a l s

per group is small, t h e t e n d e n c y for decrease ( P < 0 . 0 5 ) in s y n t h e s i s b y b o t h p a t h w a y s i n 0 2e n r i c h e d a t m o s p h e r e s is s h o w n in b o t h h a m sters a n d rat, i m p l y i n g some degree o f signific a n c e in t h e data. DISCUSSION

T h e r e are a n u m b e r o f steps i n v o l v e d in t h e de n o v o s y n t h e s i s o f t h e e x a m i n e d glycerides. T h e p r i m a r y r e a c t i o n s are 1) t h e f o r m a t i o n o f P A b y a c y l a t i o n o f sn-glycerol 3 - p h o s p h a t e ( d e r i v e d f r o m glycerol a n d a d e n o s i n e t r i p h o s p h a t e ; a n d 2) t h e h y d r o l y s i s o f PA b y p h o s p h a t a s e t o give 1,2-DG, w h i c h is a c e n t r a l interm e d i a t e for b o t h p h o s p h o l i p i d a n d triacyl-snglycerol ( T G ) b i o s y n t h e s i s . T h e 1,2-DG c a n t h e n u n i t e w i t h e i t h e r a n a c y l - C o A t o give a T G o r w i t h t h e a p p r o p r i a t e n i t r o g e n o u s base t o give a phospholipid. G l y c e r o l i n c o r p o r a t i o n i n t o lipids is a n indic a t i o n o f de n o v o lipid s y n t h e s i s , as glycerol LIPIDS, VOL. 11, NO. 7

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E.G. TOMBROPOULOS AND J.G. HADLEY

incorporation requires the formation of two ester bonds for 1,2-DG and three ester bonds for TG or phospholipids, making the initial incorporation by exchange reactions unlikely. Palmitate, on the other hand, can donate its carbons by known exchange reactions and, therefore, can label lipids b o t h through exchange reactions and through true biosynthetic processes. The ratio of (2-3H) glycerol to (1-14C) palmitate incorporation can indicate the existence of a c o m m o n precursor pool and provide an estimate of exchange reactions. The results presented here indicate that the 1,2-DG unit of PC and PE arises from a different source than the 1,2-DG of the neutral lipids. This difference in the 1,2-DG moiety between neutral and phosphollpids was observed previously with rat lung slices (18) and has been indicated by an observation of differential rates for incorporation of palmitate into neutral and phospholipids (19). Our results, suggesting that the rat and hamster lung tissue are capable of de novo PC synthesis, agree with the results obtained from newborn rabbit lung slices when the glycerol incorporation was studied (20), from the rat lung perfusion experiments when the incorporation of glucose and long chain fatty acids into lipids were examined (21), and those where the incorporation of glycerol and palmitate into lipids in vivo and in vitro by adult rabbit lung was studied (4). Studies with rat lung slices (7), on the other hand, indicated very little de novo synthesis of PC. This discrepancy could be due to different methods of tissue preparation, as it has been demonstrated that lung slices are extremely labile and easily lose certain of their b i o s y n t h e t i c capabilities: for example, the ability to synthesize fatty acids from glucose (22). It has also been shown that the ability of lung tissue to synthesize PC de novo is lost during subfractionation (5). In our studies, we include all the species of PC; it is therefore possible that our estimate does not represent an e x a c t m e a s u r e m e n t o f the surfactant dipalmitoyl-PC. The high de novo synthesis of PC found in this study is accompanied by a moderately large biosynthesis of PE. This is in keeping with the importance of the m e t h y l a t i o n p a t h w a y in PC synthesis, which would require a large pool of PE for stepwise methylation to form PC. While there was greater de novo synthesis of PE than of TG, more palmitate was incorporated into the TG. The ratio of (2-all) glycerol to (1-14C) palmitate incorporation into PC was constant during the 40 rain perfusion. This observation is in good agreement with the report of the halfLIPIDS, VOL. 11, NO. 7

life of lung PC (6). This report stated that: "The half-lives of radioactivity in the tritiuwr labled glycerol and in the 14C-labele d fatty acid portions were not significantly different in any of the four lecithin subfractions." PC can be formed from the 1,2-DG by the incorporation of a molecule o f phosphorylcholine through the cy t i d i n e m o n o ph o s p h a t e - p h o s p h o r y l c h o l i n e pathway. A second pathway for the synthesis of PC is through the stepwise methylation of PE using the methyl groups of S-adenosylmethionine. These two mechanisms of PC synthesis are usually referred to as de novo synthesis. The ratio of incorporation of methionine to choline is taken as a measurement of the relative contribution of the two mechanisms. In addition, free choline can be incorporated into PC by exchange reactions. The ratio of incorporation of (CH3-aH) methionine to (CH3-14C) choline ranged from 0.53 to 1.00. When the lung tissue was perfused with bicarbonate buffer, the contribution of the methylation pathway was greater than when it was perfused with phosphate buffer. T h e methionine:choline incorporation ratio i n d i c a t e s a s i g n i f i c a n t c o n t r i b u t i o n to PC synthesis by the methylation pathway, which agrees with our previous findings with l u n g s u b c e l l u l a r fractions (12) and with Morgan's suggestion of the importance of the m e t h y l a t i o n pathway (11). Bjr and B r e m e r ( 2 3 ) also r e p o r t e d s i g n i f i c a n t incorporation via the methylation pathway after intravenous injections in rats of the same compounds used in this study. The magnitude of synthesis via the methylation pathway in this study contrasts with that in reports on rat lung slices, in which the (CH3-14C) methionine incorporation into PC was only 2.6% of the incorporation of (CH3-14C) choline (10). The degree of utilization of methionine and choline in our study also contrasts with results from in v i v o experiments where, after intravenous administration of L-(CH3-14C) methionine and (CH3-aH) choline, 160-250 times more 3H from choline than 14C from methionine was found in lung PC (9). We believe that the perfused lung preparations allowed a more reasonable estimate to be made of the partition of the two major pathways for the formation of the nitrogenous base of PC than either lung subcellular fractions, lung slices, or whole animal experiments. This estimate may be more representative because of both the structural integrity of the perfused lung, as judged by microscopy, and the elimination of the biosynthetic activity of o t h e r organs. Inhaling an O2-enfiched atmosphere for 40 rain tends to inhibit the formation of the

LIPID SYNTHESIS BY LUNG n i t r o g e n o u s base in our p r e p a r a t i o n s . Previous e x p e r i m e n t s (24,25) f o u n d t h a t a high in vitro PO 2 i n t e r f e r e s w i t h t h e last step in t h e m e t h y l a t i o n o f PE to PC by N - m e t h y l transferase i s o l a t e d f r o m dog lung. In the p r e s e n t experim e n t s , we f o u n d b o t h a r e d u c t i o n in t h e i n c o r p o r a t i o n o f (3I-I-CH 3) m e t h i o n i n e and o f (14C-CH3) choline i n t o PC. This a p p a r e n t r e d u c t i o n in the de novo s y n t h e s i s o f PC at t h e level of the n i t r o g e n o u s bases may b e one cause o f the r e d u c e d a m o u n t s o f d i p a l m i t o y l - P C f o u n d after i n h a l a t i o n o f O 2 - e n r i c h e d atmospheres, w h i c h may result in the d e v e l o p m e n t of p u l m o n a r y distress (17,24). Also, this r e d u c t i o n c o u l d a c c o u n t for our difficulty in m a i n t a i n i n g lung preparations respiring 95% 0 2 for > 4 5 - 5 0 r r f i n ( d u e t o e d e m a ) , while similar p r e p a r a t i o n s respiring r o o m air last f o r 2-3 hr.

ACKNOWLEDGMENTS This work was done by Battelle, Pacific Northwest Laboratories, for the U.S. Atomic Energy Commission under contract AT(45-1)- 1830.

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