AMERICAN JOURNAL OF PHYSIOLOGY Printed Vol. 229, No. 5, November 1975.
in U.S.A.
Effect of ethanol
on phospholipid
metabolism
by the rat lung MI. WAGNER
AND
Cornell
Medid
Univer.uQ
H.
0.
HEINEMANN
Center,
Depurtment
of A4edicine,
York
City
K@21
faceted effects of alcohol on lipid metabolisnl raise the possibility that alcohol may also affect fatty acid utilization by the lung. The experiments to be presented demonstrate in rats that prefeeding of 40 S ethanol impairs the incorporation of palmitic acid into phospholipids of the lung L both in viva and in vitro.
WAGNER, M., AND H. 0. HEINEMANN. Effect of ethanol on phospholipid metabolism by the rat lung. Am. J. Physiol. 229(5) : 13 16-l 320. 1975.-Prefeeding of alcohol slows the in vivo incorporation of orally administered palmitic acid into phosphatidylcholine of the lung. This impairment is also demonstrable in vitro utilizing lung slices and 14C-labeled palmitate or cytidine 5’-diphospho-
choline as precursors. It is concluded that alcohol ingestion affects the utilization of precursors needed for phospholipid formation
Xew
in the lung. METHODS
alcohol;
palmitic
acid;
phosphatidylcholine
THE MAMMALIAN LUNG the synthesis of phospholipids is oriented toward the fornlation of dipalrnityl-phosphatidylcholine, a highly surface-active substance and a major component of the alveolar lining layer which serves to stabilize surface tension of the lung at varying volumes (5, 6, 13, 17, 24, 26, 30, 32, 33). This lining layer is renewed with an estimated turnover time for the lipid cornponent of approximately 14 h in the rat (1,6,3 1). The palmitic acid needed for the formation of these phospholipids may be synthesized de novo in the lung or may be derived from circulating fatty acids, some of which may originate from the intestinal tract (14, 23, 32). The phospholipid content of lung tissue and the overall turnover of lung phospholipids, measured by the incorporation of radioactively labeled precursors, is affected by the nutritional state of the animal (10, 12, 14, 28, 29). Quantitative data on the utilization by the lung of lipids absorbed from the intestinal tract are lacking, although such information would be helpful to establish interrelations between nutrition, intestinal lipid absorption, and lipid turnover in the lung. Palmitic acid enters the body from the intestinal tract via the thoracic duct predominantly in chylonlicra ( 16). The lung with its large capillary bed is the first organ to be exposed to thoracic duct lymph. It is conceivable, therefore, that the incorporation by the lung of fatty acids absorbed from the intestinal tract may be modified in situations characterized by changes in either reabsorption or utilization of these fatty acids. Alcohol excess affects the utilization of fatty acids in the liver, accelerates triglyceride synthesis by intestinal tissue, and modifies gastric and intestinal motility, intestinal lymph flow, and the absorption rate of fatty acids from the gastrointestinal tract (2, 3, 7, 18, 19, 27). These multi-
IN
Female Wistar rats weighing from 150 to 350 g were used in these experiments. All animals had free access to water at all times and followed a feeding cycle selected to promote fatty acid utilization in which they fasted for 36 h, were refed with Forrnulab by Ralston Purina Company ad libitunr for 18 h, and then fasted a second time overnight prior to the experinlent. The animals undergoing the above-mentioned feeding cycle were subdivided into two groups, one received 40 ‘,‘i ethanol by stomach tube (3.2 g/kg per day) on three successive days, the other served as the control and received equicaloric amounts of glucose in water by the same procedure. The last dose of alcohol was administered 12-15 h prior to sacrifice of the animals. Two types of experiments were perfornled with control and alcohol-fed rats : in one, living anirnals received pahnitic acid by stomach tube on the day of the study, in the other, the animals were sacrificed and slices of lung tissue were incubated in palrnitate or cytidine 5’-diphosphocholinecontaining medium. The solution given by stomach tube contained 5% albumin, 0.5 mnol pahnitic acid, and 10 PCi 14C-labeled palrnitate in a total volume of 0.6 1111. This solution was prepared by dissolving the palmitic acid first in 0.4 ml absolute alcohol. The alcohol was subsequently blown off under nitrogen with gentle heating. The administration of palmitic acid was followed by a small volume of milk (1 1111) as a wash. For the in vitro experiments, the resected organ was sliced with a &ache-Riggs tissue slicer, weighed, and incubated in 5 ml Krebs-Ringer phosphate buffer at 37°C for 2 h in a shaking water bath at 80 strokes/rnin. The Krebs-Ringer solution contained, in addition, 3 ‘1;. albunlin, 10 pmol palrnitate, 28 pm01glucose, and 0.25 &i L4C-labeled palrnitic acid. At the end of the incubation, the tissue slices were separated from the medium, washed with 5 ml KrebsRinger solution containing 3 ‘;;. albumin for removal of
1316 Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (142.103.160.110) on January 2, 2019.
PHOSPHOLIPID
METABOLISM
BY
RAT
LUNG
loosely attached fatty acids, and extracted in chloroformmethanol. In another set of in vitro experiments, lung tissue slices were incubated in a medium in which the 14C-labeled palmitic acid was substituted by 0.25 &i 14C-labeled cytidine 5’-diphosphocholine. The experimental period lasted from 2 to 6 h in the in viva experiments and 2 h in the in vitro studies. The animals were sacrificed under light ether anesthesia, blood was drawn, and lung, liver, and intestine were resected, weighed, and immediately extracted with 2: 1 chloroform-methanol. The lipids were extracted by the method of Folch et al. and after removal by suction of the upper aqueous phase, were reduced to dryness under nitrogen and weighed to a constant weight. For recovery of remaining 14C activity, the intestinal tract was removed in toto after ligatures were placed around the esophagus and lower portion of the large bowel. The mesentery and the associated lymphatics were dissected and discarded to eliminate lipids which had traversed the intestinal mucosa. Liver tissue was removed for determination of lipid weight which was used as the criterion for alcohol-induced changes in tissue metabolism. Control values ranged from 26.4 to 35.2 mg/g wet wt in 250- to 350-g rats and 24.5-30.0 mg/g wet wt in 150- to 250-g rats. Lipid weight increased ‘in all alcohol-fed animals from 8.7 to 16.8 mg/g (P < .Ol). The extracted lipids were separated into neutral lipids and phospholipids on columns using 2 g of silicic acid. The two classes of lipids were eluted with 30 1111 of chloroform followed by th.e same volume of methanol. Each fraction was reduced to dryness, dissolved in a known volume of chloroform-methanol, and aliquots were taken for total radioactivity, phosphorus, and for separation in to individual lipids by thin-layer chromatography (TLC) using silica gel-H. Radioactivity was determined by liquid scintillation spectrophotometry with an efficiency of 92 c/i. Phospholipids were separated by the solvent scheme: chloroform 80, methanol 25, water 4, glacial acetic acid 1; neutral lipids were separated using hexane 70, diethyl ether 30, glacial acetic acid 2. Following visualization with iodine vapor and identification by comparison with pure standards (Applied Science Laboratories, Inc.), the individual spots were scraped directly into scintillation vials and counted with 4 ‘/‘; Cab-O-S‘1 1 in scintillation fluid. A second aliquot was separated by the same TLC scheme for the identification of the major phospholipids. The individual spots were scraped and eluted using 5 ‘,‘; HCI-methanol for phosphorus determination by the modified method of Marinetti (21). All results are expressed as counts per minute per milligram total lipid or microgram phospholipid phosphorus. RESULTS
The
results
are summarized
in Table
1 and Figs.
1 and
2. In Vivo Experiments
with Normnl
Animuls
Intestinal tract. The absorption of palmitic acid reflected by the average disappearance of 14C activity from the intestinal tract varied between individual observations. There
50
i i !t I
I
1
1
2
I
I
3 Hou t-s
4
,
1
5
6
FIG. 1. Absorption of palmitic acid as reflected by recovery of 14C activity in intestinal tract 2-6 h after oral administration of Wlabeled palmitic acid. There is a significant inverse correlation (I = -0.80) between recovery of palmitic acid and duration of observation period.
was, nevertheless, a significant inverse correlation (r = - 0.80) between the recovery of administered palmitate (2 X lo7 counts/min [ 1-14C]palmitic acid equivalent to 0.5 mm01 of fattv acid) and the duration of the experimental period which ranged from 2 to 6 h (Fig. 1). On the average 10 It 2 :I of the administered dose was recovered in the intestinal tract 6 h after administration of palmitic acid. Lung. Total radioactivity recovered in lung tissue increased with time to reach 867 It 5 16 counts/min per mg extractable lipid at 6 h, reflecting 0.022 ,umol of palmitate incorporation per milligram tissue lipid (Table 1). The 14C activity in the phospholipid fraction represented 85 5; of total recovered radioactivity at 6 h, reaching 45 & 26 counts/min per 7 phosphorus in phosphatidylcholine (PC>. In Vivo Experiments
with Ethanol-Fed
Rats
The animals were studied after 3 days of ethanol feeding and sacrificed 6 h after receiving an oral load of palmitic acid. The increased lipid content of liver tissue (see METHODS) was used as evidence for ethanol-induced changes in tissue metabolism (2, 8, 11, 20). Intestinal tract. Recovery of the administered 14C activity in the intestinal tract was 36 & 5 C;T compared to 10 =t 2 % fpr the control group (p < 0.001) (Fig. 2). Lung. The radioactivity recovered in lung tissue was less than in the control group and averaged 343 zfr 201 counts/ min per mg extractable lipid (Table 1). The chance that this difference was spurious was less than 5 5. The major portion of the radioactivity was recovered in the phospholipid moiety (80 =t 10%). The phosphorus content of extracted phospholipids was statistically not different from control (19 vs. 23 y/mg total lipid; Table 1), but 14C activity per microgram phospholipid phosphorus in both
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (142.103.160.110) on January 2, 2019.
1318 TABLE
M.
1. Effect of ethanol on lung lipid metabolism .____n
Lung
Protocol
A*
C* - ---- __---
Lipid
Weight
1°C Activity Total
______I___
C
--________--~-_
mg/g wet wt ~__
A
-
C
A
[I-W]palmitic
In vivot
9
6
29.1zk3.6
32.2zk3.4
23zk6
NS In vitro
6
6
20.7A.9
20.6rt2.2
6
5
18.5zt2.4
19zt3
20&2
20.5zt3.5
18zk2
Lung slices of control and alcohol-fed animals were incubated in an in vitro system with either 14C-labeled palmitic acid or 14C-labeled cytidine 5’-diphosphocholine as a precursor to verify the changes observed in alcoholloaded living animals. Incubation with 14C-labeled palmitic acid (Table 1). In control animals 14C activity in extractable tissue lipids was 7,060 + 1,485 counts/min per mg reflecting incorporation of 0.16 ,umol of palmitic acid; in ethanol-fed rats 14C activity was 6,125 & 1,669 counts/min per mg lipid representing 0.14 pmol of palmitic acid (statistically not different from control). The radioactivity recovered in the total phospholipid fraction of alcohol-fed rats was less than control (99 vs. 116 counts/min per y phosphorus), but the difference was statistically not significant. However, in phosphatidylcholine r
40 I I
z 30 .-.c, t -c6 &xl t! 2 10
T
Control
(9)
Alcohol
(6)
p< am 2. Recovery of 14C activity administration of labeled palmitic is statistically significant. FIG.
in intestinal tract 6 h after oral acid. Difference between 2 groups
_~
(PL)
34*19
Phosphatidylcholine$ C per 7 P
16zk9
___A
(PC)
.^
_I---.
-
6,125&1,669
116zk16
17&4
45=t26
< .05
6,796&679
in U.S.A.
Effect of ethanol
on phospholipid
metabolism
by the rat lung MI. WAGNER
AND
Cornell
Medid
Univer.uQ
H.
0.
HEINEMANN
Center,
Depurtment
of A4edicine,
York
City
K@21
faceted effects of alcohol on lipid metabolisnl raise the possibility that alcohol may also affect fatty acid utilization by the lung. The experiments to be presented demonstrate in rats that prefeeding of 40 S ethanol impairs the incorporation of palmitic acid into phospholipids of the lung L both in viva and in vitro.
WAGNER, M., AND H. 0. HEINEMANN. Effect of ethanol on phospholipid metabolism by the rat lung. Am. J. Physiol. 229(5) : 13 16-l 320. 1975.-Prefeeding of alcohol slows the in vivo incorporation of orally administered palmitic acid into phosphatidylcholine of the lung. This impairment is also demonstrable in vitro utilizing lung slices and 14C-labeled palmitate or cytidine 5’-diphospho-
choline as precursors. It is concluded that alcohol ingestion affects the utilization of precursors needed for phospholipid formation
Xew
in the lung. METHODS
alcohol;
palmitic
acid;
phosphatidylcholine
THE MAMMALIAN LUNG the synthesis of phospholipids is oriented toward the fornlation of dipalrnityl-phosphatidylcholine, a highly surface-active substance and a major component of the alveolar lining layer which serves to stabilize surface tension of the lung at varying volumes (5, 6, 13, 17, 24, 26, 30, 32, 33). This lining layer is renewed with an estimated turnover time for the lipid cornponent of approximately 14 h in the rat (1,6,3 1). The palmitic acid needed for the formation of these phospholipids may be synthesized de novo in the lung or may be derived from circulating fatty acids, some of which may originate from the intestinal tract (14, 23, 32). The phospholipid content of lung tissue and the overall turnover of lung phospholipids, measured by the incorporation of radioactively labeled precursors, is affected by the nutritional state of the animal (10, 12, 14, 28, 29). Quantitative data on the utilization by the lung of lipids absorbed from the intestinal tract are lacking, although such information would be helpful to establish interrelations between nutrition, intestinal lipid absorption, and lipid turnover in the lung. Palmitic acid enters the body from the intestinal tract via the thoracic duct predominantly in chylonlicra ( 16). The lung with its large capillary bed is the first organ to be exposed to thoracic duct lymph. It is conceivable, therefore, that the incorporation by the lung of fatty acids absorbed from the intestinal tract may be modified in situations characterized by changes in either reabsorption or utilization of these fatty acids. Alcohol excess affects the utilization of fatty acids in the liver, accelerates triglyceride synthesis by intestinal tissue, and modifies gastric and intestinal motility, intestinal lymph flow, and the absorption rate of fatty acids from the gastrointestinal tract (2, 3, 7, 18, 19, 27). These multi-
IN
Female Wistar rats weighing from 150 to 350 g were used in these experiments. All animals had free access to water at all times and followed a feeding cycle selected to promote fatty acid utilization in which they fasted for 36 h, were refed with Forrnulab by Ralston Purina Company ad libitunr for 18 h, and then fasted a second time overnight prior to the experinlent. The animals undergoing the above-mentioned feeding cycle were subdivided into two groups, one received 40 ‘,‘i ethanol by stomach tube (3.2 g/kg per day) on three successive days, the other served as the control and received equicaloric amounts of glucose in water by the same procedure. The last dose of alcohol was administered 12-15 h prior to sacrifice of the animals. Two types of experiments were perfornled with control and alcohol-fed rats : in one, living anirnals received pahnitic acid by stomach tube on the day of the study, in the other, the animals were sacrificed and slices of lung tissue were incubated in palrnitate or cytidine 5’-diphosphocholinecontaining medium. The solution given by stomach tube contained 5% albumin, 0.5 mnol pahnitic acid, and 10 PCi 14C-labeled palrnitate in a total volume of 0.6 1111. This solution was prepared by dissolving the palmitic acid first in 0.4 ml absolute alcohol. The alcohol was subsequently blown off under nitrogen with gentle heating. The administration of palmitic acid was followed by a small volume of milk (1 1111) as a wash. For the in vitro experiments, the resected organ was sliced with a &ache-Riggs tissue slicer, weighed, and incubated in 5 ml Krebs-Ringer phosphate buffer at 37°C for 2 h in a shaking water bath at 80 strokes/rnin. The Krebs-Ringer solution contained, in addition, 3 ‘1;. albunlin, 10 pmol palrnitate, 28 pm01glucose, and 0.25 &i L4C-labeled palrnitic acid. At the end of the incubation, the tissue slices were separated from the medium, washed with 5 ml KrebsRinger solution containing 3 ‘;;. albumin for removal of
1316 Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (142.103.160.110) on January 2, 2019.
PHOSPHOLIPID
METABOLISM
BY
RAT
LUNG
loosely attached fatty acids, and extracted in chloroformmethanol. In another set of in vitro experiments, lung tissue slices were incubated in a medium in which the 14C-labeled palmitic acid was substituted by 0.25 &i 14C-labeled cytidine 5’-diphosphocholine. The experimental period lasted from 2 to 6 h in the in viva experiments and 2 h in the in vitro studies. The animals were sacrificed under light ether anesthesia, blood was drawn, and lung, liver, and intestine were resected, weighed, and immediately extracted with 2: 1 chloroform-methanol. The lipids were extracted by the method of Folch et al. and after removal by suction of the upper aqueous phase, were reduced to dryness under nitrogen and weighed to a constant weight. For recovery of remaining 14C activity, the intestinal tract was removed in toto after ligatures were placed around the esophagus and lower portion of the large bowel. The mesentery and the associated lymphatics were dissected and discarded to eliminate lipids which had traversed the intestinal mucosa. Liver tissue was removed for determination of lipid weight which was used as the criterion for alcohol-induced changes in tissue metabolism. Control values ranged from 26.4 to 35.2 mg/g wet wt in 250- to 350-g rats and 24.5-30.0 mg/g wet wt in 150- to 250-g rats. Lipid weight increased ‘in all alcohol-fed animals from 8.7 to 16.8 mg/g (P < .Ol). The extracted lipids were separated into neutral lipids and phospholipids on columns using 2 g of silicic acid. The two classes of lipids were eluted with 30 1111 of chloroform followed by th.e same volume of methanol. Each fraction was reduced to dryness, dissolved in a known volume of chloroform-methanol, and aliquots were taken for total radioactivity, phosphorus, and for separation in to individual lipids by thin-layer chromatography (TLC) using silica gel-H. Radioactivity was determined by liquid scintillation spectrophotometry with an efficiency of 92 c/i. Phospholipids were separated by the solvent scheme: chloroform 80, methanol 25, water 4, glacial acetic acid 1; neutral lipids were separated using hexane 70, diethyl ether 30, glacial acetic acid 2. Following visualization with iodine vapor and identification by comparison with pure standards (Applied Science Laboratories, Inc.), the individual spots were scraped directly into scintillation vials and counted with 4 ‘/‘; Cab-O-S‘1 1 in scintillation fluid. A second aliquot was separated by the same TLC scheme for the identification of the major phospholipids. The individual spots were scraped and eluted using 5 ‘,‘; HCI-methanol for phosphorus determination by the modified method of Marinetti (21). All results are expressed as counts per minute per milligram total lipid or microgram phospholipid phosphorus. RESULTS
The
results
are summarized
in Table
1 and Figs.
1 and
2. In Vivo Experiments
with Normnl
Animuls
Intestinal tract. The absorption of palmitic acid reflected by the average disappearance of 14C activity from the intestinal tract varied between individual observations. There
50
i i !t I
I
1
1
2
I
I
3 Hou t-s
4
,
1
5
6
FIG. 1. Absorption of palmitic acid as reflected by recovery of 14C activity in intestinal tract 2-6 h after oral administration of Wlabeled palmitic acid. There is a significant inverse correlation (I = -0.80) between recovery of palmitic acid and duration of observation period.
was, nevertheless, a significant inverse correlation (r = - 0.80) between the recovery of administered palmitate (2 X lo7 counts/min [ 1-14C]palmitic acid equivalent to 0.5 mm01 of fattv acid) and the duration of the experimental period which ranged from 2 to 6 h (Fig. 1). On the average 10 It 2 :I of the administered dose was recovered in the intestinal tract 6 h after administration of palmitic acid. Lung. Total radioactivity recovered in lung tissue increased with time to reach 867 It 5 16 counts/min per mg extractable lipid at 6 h, reflecting 0.022 ,umol of palmitate incorporation per milligram tissue lipid (Table 1). The 14C activity in the phospholipid fraction represented 85 5; of total recovered radioactivity at 6 h, reaching 45 & 26 counts/min per 7 phosphorus in phosphatidylcholine (PC>. In Vivo Experiments
with Ethanol-Fed
Rats
The animals were studied after 3 days of ethanol feeding and sacrificed 6 h after receiving an oral load of palmitic acid. The increased lipid content of liver tissue (see METHODS) was used as evidence for ethanol-induced changes in tissue metabolism (2, 8, 11, 20). Intestinal tract. Recovery of the administered 14C activity in the intestinal tract was 36 & 5 C;T compared to 10 =t 2 % fpr the control group (p < 0.001) (Fig. 2). Lung. The radioactivity recovered in lung tissue was less than in the control group and averaged 343 zfr 201 counts/ min per mg extractable lipid (Table 1). The chance that this difference was spurious was less than 5 5. The major portion of the radioactivity was recovered in the phospholipid moiety (80 =t 10%). The phosphorus content of extracted phospholipids was statistically not different from control (19 vs. 23 y/mg total lipid; Table 1), but 14C activity per microgram phospholipid phosphorus in both
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (142.103.160.110) on January 2, 2019.
1318 TABLE
M.
1. Effect of ethanol on lung lipid metabolism .____n
Lung
Protocol
A*
C* - ---- __---
Lipid
Weight
1°C Activity Total
______I___
C
--________--~-_
mg/g wet wt ~__
A
-
C
A
[I-W]palmitic
In vivot
9
6
29.1zk3.6
32.2zk3.4
23zk6
NS In vitro
6
6
20.7A.9
20.6rt2.2
6
5
18.5zt2.4
19zt3
20&2
20.5zt3.5
18zk2
Lung slices of control and alcohol-fed animals were incubated in an in vitro system with either 14C-labeled palmitic acid or 14C-labeled cytidine 5’-diphosphocholine as a precursor to verify the changes observed in alcoholloaded living animals. Incubation with 14C-labeled palmitic acid (Table 1). In control animals 14C activity in extractable tissue lipids was 7,060 + 1,485 counts/min per mg reflecting incorporation of 0.16 ,umol of palmitic acid; in ethanol-fed rats 14C activity was 6,125 & 1,669 counts/min per mg lipid representing 0.14 pmol of palmitic acid (statistically not different from control). The radioactivity recovered in the total phospholipid fraction of alcohol-fed rats was less than control (99 vs. 116 counts/min per y phosphorus), but the difference was statistically not significant. However, in phosphatidylcholine r
40 I I
z 30 .-.c, t -c6 &xl t! 2 10
T
Control
(9)
Alcohol
(6)
p< am 2. Recovery of 14C activity administration of labeled palmitic is statistically significant. FIG.
in intestinal tract 6 h after oral acid. Difference between 2 groups
_~
(PL)
34*19
Phosphatidylcholine$ C per 7 P
16zk9
___A
(PC)
.^
_I---.
-
6,125&1,669
116zk16
17&4
45=t26
< .05
6,796&679
