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Effect of ethanol consumption on the phospholipid composition of rat liver microsomes and mitochondria
Biochimica et Biophysics Acta, 712 (1982) 225-233 Elsevier Biomedical Press
225
BBA 5 1142
EFFECT OF ETHANOL CONSUMPTION ON THE PHOSPHOLIPID LIVER MICROSOMES AND MITOCHONDRIA CAROL
C. CUNNINGHAM,
STEPHEN
FILUS,
RALPH
E. BOITENUS
and PRISCILLA
COMPOSITION
OF RAT
I. SPACH
Department of Biochemistry Bowman Gray School of Medicine, Wake Forest Unioersity, Winston-Salem, NC 27103 (U.S.A.) (Received
January
26th, 1982)
Key words: Ethanol; Microsome; Mitochondria; Phospholipid; (Rat liver)
Male Sprague-Dawley rats were maintained for 31 days on a liquid diet containing 36% of calories as ethanol. Pair-fed controls were administered a similar diet, but with maltose-dextrin isocalorically substituted for ethanol. A phospholipid analysis has been carried out in liver microsomes and mitochondria isolated from the two groups of animals. The phospholipid phosphorus/protein ratio was not significantly different in the organelles of the ethanol-fed animals as compared to the same organelles of liquid diet controls, which indicates that ethanol feeding did not influence the total phospholipid content of microsomes and mitochondria. The phospholipid distribution within organelles was not changed, except for a significant increase in the phosphatidylinositol content of microsomes from ethanol-fed animals. The fatty acid compositions of both microsomal and mitochondrial phospholipids were significantly altered by ethanol feeding. In microsomes from ethanol-fed rats, palmitic acid levels were lowered in the total phospholipid fraction, phosphatidylcholine and phosphatidylethanolamine; oleic acid levels were elevated in microsomal phosphatidylethanolamine. In mitochondria from ethanol-fed animals, palmitic and arachidonic acid were lowered in phosphatidylcholine and phosphatidylethanolamine. Oleic and linoleic acid were elevated in the same phospholipids. In contrast, IhroIeic acid levels in cardiolipin were depressed significantly. These alterations in the fatty acid composition are suggestive of ethanol-induced changes in fatty acid desaturation activities.
Introduction
[6,7] activities may be linked, at least in part, to ethanol-induced changes in membrane phospholipids. It would appear, however, that changes observed in those activities associated with the oxidative phosphorylation system cannot be linked with changes in the fatty acid composition of bulk-phase phospholipids from mitochondrial membranes. In an earlier study [3] we compared the energy-linked properties and associated enzyme activities, and the fatty acid composition of the total phospholipids in mitochondria from rats fed either chow or a control liquid diet, or an ethanol-containing liquid diet. Few differences between the fatty acid compositions of liver mitochondria phospholipids from liquid diet controls and liquid diet ethanol-fed
Several studies have demonstrated that the oxidative phosphorylation system in rat liver mitochondria is adversely affected by chronic ethanol consumption [l-3]. The oxidative phosphorylation system, being membrane-associated, requires phospholipids for its proper functioning. Moreover, those enzymes comprising the oxidative phosphorylation system require phospholipids for expression of their activities [4,5]. Recent studies suggest that changes related to ethanol feeding in both the mitochondrial respiratory [6] and ATPase Abbreviations: PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine. OOOS-2760/82/0000-0000/$02.75
0 1982 Elsevier Biomedical
Press
226
animals were seen, whereas several significant changes in energy-linked functions and associated enzyme activities were noted. In contrast, when chow-fed and liquid diet control animals were compared, dramatic differences in the fatty acid composition of mitochondrial phospholipids were noted, but no differences were observed in either energy-linked functions or associated enzyme activities. The above observations tend to discount that changes in the fatty acid composition of bulk phase lipids are responsible for ethanol-induced alterations in mitochondrial associated energy-linked functions. They did not eliminate the possibility, however, that alterations in either the concentration or the fatty acid composition of a particular phospholipid could be responsible for some of the ethanol-induced alterations in the oxidative phosphorylation system. In this communication we report the concentrations of mitochondrial phospholipid classes isolated from the livers of ethanol-fed rats. In addition, the fatty acid composition of each mitochondrial phospholipid was measured, with the ethanol-induced changes being reported in this paper. The changes in fatty acid composition demonstrate the possibility that alterations in individual phospholipids could result in changes in the activity of membrane-associated enzymes, since a particular phospholipid, rather than the total phospholipid complement, might serve as a boundary lipid for an enzyme whose activity was altered by ethanol feeding. In addition to measuring the ethanol-induced alterations in mitochondrial phospholipid composition, we have also determined those changes in phospholipids which occur in microsomes. Since the endoplasmic reticulum is the site of synthesis for most mitochondrial phospholipids, a comparison of the ethanol-induced alterations in phospholipids from both organelles provides us with information that suggests the origin of the changes in the mitochondrial phospholipids. Experimental procedures Materials Sprague-Dawley rats were obtained from Charles River Breeding Laboratories, North Wilmington, MA. Lieber/DeCarli liquid diets were
obtained from Bio-Serv, Frenchtown, NJ. Phospholipid standards were purchased from Serdary Research Laboratories, London, Ontario, Canada. Other reagents, including supplies for fatty acid analyses, were obtained as listed in Spach et al. [3]. Methods Diets, and preparation of mitochondria and microsomes. Male Sprague-Dawley rats weighing 150-250 g were fed for 31 days on a nutritionally adequate liquid diet [8] in which ethanol provided 36% of the total calories. Pair-fed control rats received the same diet, but with maltose-dextrin isocalorically substituted for ethanol. Preparation of tightly coupled mitochondria and protein determinations were carried out as described previously [3]. Microsomes were prepared from the postmitochondrial (8700 X g) supernatant by the following procedure. The supernatant was centrifuged at 17300 X g for 10 min to remove lysosomes and then at 78500 X g for 90 min to sediment the microsomes. The microsomes were resuspended in 40 ml of 0.25 M sucrose, pH 7.4, and recentrifuged at 78 500 X g for 90 min. The washed microsomes were resuspended in 10 ml of 0.25 M sucrose, pH 7.4. Initial experiments measuring marker enzymes demonstrated that the microsomes were minimally contaminated with other cell components. Values obtained for NADPHcytochrome c reductase activity indicate that the microsomal fraction is enriched by as much as 22-fold when compared to mitochondrial preparations (microsomes, 67 nmol/min per mg protein; mitochondria, 3 nmol/min per mg protein). Phospholipid analyses of mitochondria and microsomes. Lipids were extracted from cell organelles immediately after their preparation by the procedure of Bligh and Dyer [9] to minimize hydrolysis by phospholipases, and aliquots were taken for lipid phosphorus analyses [IO]. Either 30 mg mitochondrial protein or 16 mg microsomal protein were extracted to obtain the lipid complement. Phospholipids within the extract were fractionated by two-dimensional thin-layer chromatography on silica gel H. The chromatography plate was developed in chloroform/acetone,/ methanol/acetic acid/water (10 : 4 : 2 : 3 : 1, V/V) and stored overnight in a nitrogen atmosphere.
227
ages rt S.E. for the numbers indicated. Statistical analyses were performed using the t test for two means [ 111.
The plate was then developed in a second dimension with butanol/acetic acid/water (80: 26 : 26, v/v). This combination of solvents provided good separation of all phospholipid classes, including phosphatidylserine (PS) and phosphatidylinositol (PI). To locate the phospholipids, the chromatogram was sprayed lightly with dichlorofluorescein in 95% ethanol and then illuminated with an ultraviolet lamp. The phospholipid spots, identified on the chromatogram by use of purified standards, were scraped from the plate. The phospholipids were eluted from silicic acid by repeated washing with chloroform/methanol/water (1: 2 : 0.2, v/v). This extraction procedure is quantitative for all phospholipids except sphingomyelin; the recovery is 80-85% with the latter (King, L. and Waite, M., personal communication). The phospholipid extracts were analyzed for lipid phosphorus levels and for fatty acid composition as outlined previously [3]. The fatty acids were identified by comparing their elution times with those of purified standards utilizing a Varian 3700 gas chromatograph. Quantitation was carried out with a Varian CDS-1 11 data processor. The values reported in the tables are the averTABLE
Results
The livers from animals fed the ethanol-containing diets demonstrated fatty liver, as assessed by their increased size and their excess lipid. Table I demonstrates that the phospholipid distribution in microsomes and ~t~hondria isolated from these livers was minimally affected by ethanol feeding. Of all the phospholipids in both organelles, only microsomal PI was altered as a resuit of the ethanol diet. This metabolically important phospholipid was increased 66% in microsomes, but its level in mitochondria was not affected in the ethanol-fed animals. Cardiolipin was lowered in liver microsomes from ethanol-fed rats, which suggested that ethanol microsomes were less contaminated with mit~hondria since c~diolipin is felt to be localized to the latter organelle. The phospholipid-toprotein ratio was not altered in either liver microsomes or mitochondria as a result of ethanol feed-
I
PHOSPHOLIPID CALLY
DISTRIBUTION
IN LIVER
MICROSOMES
AND
MITOCHONDRIA
OF RATS FED ETHANOL
CHRONI-
For microsomes, n = 8 control and 9 ethanol preparations. Total phospholipid: control, 441 -t 24, nmol phospholipid phosphorus/mg protein; ethanol, 481.227 nmol phospholipid phosphorus/mg protein. For mitochondria, n = 11 control preparations and 7 ethanol preparations. Total phospholipid: control, 184% 10 nmol phospholipid phosphorus/mg protein; ethanol, 19Ok9 nmol phospholipid phospho~s/mg protein. Ail values are mean* SE. Phospholipid
type
Distribution
(mol’k;)
Microsomes
Phosphatidybholine Lysophosphatidylcholine Phosphatidylethanolamine Lysophosphatidylethanolamine Phosphatidylinositol Phosphatidyise~ne Sphingomyehn ’ Cardiolipin ’ Significantly
different different
Control
Ethanol
Control
Ethanol
66.0 k1.5 1.01*0.27 23.1 =I.4 0.7520.22 3.3620.64 2.38 2 0.68 2.6lt0.30 0.797tO.17
64.4 -t 1.8 1.36f0.23 23.0 2 1.2 1.18-‘0.46 5.58*0.51 a I .94 * 0.27 2.19-0.33 0.33 5 0.09 =
48.8 12.1 o&=0.19 38.8 - 1.7 0.44*0.16 1.89kO.31 0.67f0.16 0.63-0.16 8.2 kO.90
53.0 * 1.7 0.54~0.16 34.6 * 1.8 0.42-tO.18 2.2920.30 0.98 kO.26 0.63CO.15 7.54+0.71
from control (P ~0.025). may be as much as 20% too low due to difficulties from control (P ~0.05).
b The values for sphingomyel~n f Significantly
Mit~hondria
with its extraction
from silicic acid.
TABLE
II
FATTY
ACID
DISTRIBUTION
The data were obtained Phosphohpid
FOR MICROSOMAL
from microsomal
type
PHOSPHOLIPIDS
preparations
isolated
from nine pairs of pair-fed
Animal
Fatty
acid distribution
animals.
Values are meant-
S.E.
(mol%)
group 16:O
Phosphatidylchohne
16:l
18:O
2.2-cO.4 1.8i0.2
23.2kO.7 24.5 -c 0.8
Control Ethanol-fed
22.5 to.9 16.9-0.6
Lysophosphatidylcholine
Control Ethanol-fed
28.8‘4.0 29.1-t 1.8
4.7= 1.2 4.420.3
28.9* 3.5 30.5 * 1.7
Phosphatidylethanolamine
Control Ethanol-fed
21.9’-2.1 16.1) 1.1 *
1.4kO.2 1.110.3
31.4t3.0 27.8r2.0
Lysophosphatidylethanolamine
Control Ethanol-fed
25.31 1.8 26.6 rt 2.2
5.4* 1.0 7.02 I.0
23.71-2.8 24.9k2.3
Phosphatidylinositol
Control
***
9.62 1.4 6.5fl.l *
Ethanol-fed
2.220.5 I.0f0.2
44.5 * 4.0 45.2-c 1.8
+
Phosphatidylserine
Control Ethanol-fed
16.2’1.9 15.2-c 1.7
3.5 -CO.6 3.950.8
38.1 “3.6 35.7k3.6
Sphingomyelin
Control Ethanol-fed
30.5 * 3.0 30.422.8
4.0-t0.8 5.350.9
13.92 1.7 16.6~ 1.8
Total phosphohpid
Control Ethanol-fed
17.5’00.6 13.8kO.4
1.2’0.4 0.6iO.l
23.3* 1.0 22.6 -c 2.6
a The predominant fatty acids were 22: 5 (control, * P (0.05; **p co.01; ***p
III
FATTY
ACID
DISTRIBUTION
The numbers in parentheses *** P ~0.001. Phosphohpid
6.1 mol%; ethanol,
FOR MITOCHONDRIAL
indicate
the number
8.5 mol%) and 24: 0 (control,
5.1 mol%; ethanol,
3.6 mol%).
PHOSPHOLIPIDS
of pairs of animals
Animal
type
***
used for analyses.
Fatty
Values are meani
acid distribution
S.E. * P (0.05;
** P
(mol%)
group 16:0
Phosphatidylcholine
Control Ethanol-fed
(20)
19.9kO.6 16.6kO.6
16:
***
I
18:O
1.9kO.2 1.9*0.1
20.4-tO.6 23.1 50.8
5.5% 1.5 7.2k2.3
14.3k2.1 17.0 ‘- 3.0
1.3r0.2 1.6*0.2
26.5~ 25.9i
Control Ethanol-fed
31.9k4.5 27.41- 2.5
Control Ethanol-fed
17.9*0.8 15.5*0.9
Control Ethanol-fed
30.9k4.8 32.3k4.3
6.5 kO.9 6.1 ‘2.3
16.8& 1.9 13.0*4.0
Phosphatidylinositol(20)
Control Ethanol-fed
16.0+2.0 17.6 2 2.4
4.4kO.8 5.3% 1.3
30.1 k2.3 30.9* 3.2
Phosphatidylserine
Control Ethanol-fed
26.1* 2.6 20.4k2.8
4.2kO.6 3.2’1.0
23.2k2.9 21.2’4.1
Control Ethanol-fed
7.1 eo.7 8.5-‘1.1
2.9kO.4 2.41-0.3
Lysophosphatidylchohne Phosphatidylethanolamine
Lysophosphatidylethanolamine
Cardiohpin
(17)
(11)
(8)
(19) (8)
*
1.2 1.9
2.2kO.3 3.41-0.6
**
229
18:l
18:2
20:3
20:4
2216
20:5, 22:4 2215, 24:0,
Unidentified
24:l 11.4*0.7
1.1*0.1
2.11-0.4 2.OkO.2
2.920.4
2.120.6
19.9k2.8 22.9-c 1.3
1.2-t0.3
10.8kO.5
1.5kO.2
4.2-t0.8
4.1 -to.9 3.6-cO.6
1.1 eo.3 1.320.5
3.4* 1.2 2.6kO.7
3.4-0.7 2.O-tO.6
5.622.5 5.82 1.2
7.0* 3.6 6.1k2.2
5.5 kO.6 6.5kO.3
0.4kO.1 1.82 1.0
4.62 1.0 4.3eo.5
3.1 to.7 2.3-t0.5
3.820.5 6.4* 1.8
19.2% 1.8 15.Ok2.3
4.7% 1.5 4.2-t 1.1
2.121.6 2.1k1.2
3.8kO.9 2.OeO.7
6.4* 1.5 7.6* 1.9
7.722.1 4.7* 1.3
7.1’-1.0 7.5eo.4
6.024.1 2.5 eO.3
0.7kO.2 3.o-co.9
22.1e3.8 25.1 c2.5
1.5-0.4 1.9*0.5
3.3f0.9 4.02 1.0
2.3% 1.0 3.2kO.8
12.3kl.6 11.0r1.3
3.3 kO.7 3.2% 1.1
1.4-0.9 0.9co.4
13.4k3.1 12.6k2.1
3.5kO.7 3.720.5
4.6-f 1.0 5.121.3
3.6” 1.2 8.7~3.2
14.3k2.5 12.4* 1.8
3.1 eo.7 4.OkO.3
I.32 1.0 0.6f0.3
1.3 1.5
4.1%1.1 3.020.9
Il.35 1.8 a 12.8k3.4
14.2k3.9 10.9” 1.6
9.6eo.7 11.0*0.4
8.1 e-o.5 8.5 eO.4
0.6-tO.2 1.4kO.l
27.6* 1.5 28.6kO.5
5.2kO.3 4.5kO.6
13.3kO.7 13.5*0.4 13.1k1.2 14.3-c 1.5 11.0*0.6 13.OkO.7
*
18:l
11.0*0.3 13.1kO.6
18:2
**
15.Ok2.4 18.Ok2.5
8.4kO.3 9.820.4
17.Ok3.6 20.622.4 1.8t- 1.0 4.2t-2.1
*
3.3a 4.oc
**
20:3
**
0.620.1 1.5kO.l 0.7-to.3 0.420.4
10.1 k2.2 4.9-cO.8
0.4kO.1 0.8 r0.2
20:4
***
22~6
29.6’0.9 25.2kl.l
**
3.5* 1.8 4.3*2.2
3.3eO.2 4.1kO.2
4.OkO.8 3.1t1.1
4.2-t 1.1 10.723.9
7.6* 1.1 12.Ok3.6
6.6kO.6 5.8 20.6
2.5k1.3 3.5e1.1
3.7kO.3 5.810.7
6.8-1.9 14.7k6.2
7.1k2.1 14.2k4.8
14.62 1.8 10.4% 1.1
5.7a2.5 2.3-cO.9
1.420.6 0.9eo.5
1.6kO.7 3.9k3.0
7.723.8 2.3k1.1
11.3-cO.8 11.8-1.1
2.8eO.5 3.1 kO.5
1.3kO.3 1.2~0.4
18.4k2.0 14.3k2.1
3.9t0.8 2.0-t0.4
16.822.8 10.3* 1.4
3.3-cO.6 4.4* 2.2
1.1*0.3 3.7e3.5
5.9-2.1 4.8* 1.8
2.020.2 5.2k2.3
2.420.3 1.8kO.S
51.3k2.0 42.8k3.9
*
1.3 1.7 *
Unidentified
1.5eO.2 1.7kO.3
4.120.2 5.950.8
24.OkO.8 23.2) 1.4
26.72 21.9*
20:5, 22~4, 22:5, 24:0 24: 1
4.2-cO.9 6.7*2.7
3.4*0.3 3.OkO.3
10.3kO.4 13.3% 1.3 *
*
2.6-0.7 2.3f0.6
6.OkO.8 5.6kl.O
5.OkO.7 8.0* 1.7
4.822.1 2.3-cO.7
7.9* 1.5 17.6k7.5
6.7* 1.3 11.9k3.5
1.7-co.3 1.3*0.2
1.8kO.3 4.0 * 2.0
*
**
**
4.6kO.6 7.4* I.2 *
230 TABLE
IV
ETHANOL-INDUCED PHOSPHOLIPIDS
ALTERATIONS
IN PARAMETERS
RELATED
TO FATTY
ACID
COMPOSITION
OF MEMBRANE
The data on microsomal lipids were obtained from nine pairs of pair-fed animals. The data for mitochondrial phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol were obtained from 20, 19 and 19 pairs of pair-fed animals respectively. The values were obtained by first dividing the mol% value for oleic acid by that of palmitic acid and then calculating the average-t S.E. The C20:4/C18:2 ratio was obtained in like manner. C, phospholipid from control animals; E, phospholipid from ethanol-fed animals.
Organelle
Phospholipid
Microsomes
Total, C Total, E
C18:
P Phosphatidylcholine, Phosphatidylcholine,
C E
P Phosphatidylethanolamine, Phosphatidylethanolamine,
C E
P Phosphatidylinositol, Phosphatidylinositol,
C E
P Mitochondria
Phosphatidylcholine, Phosphatidylcholine,
C E
P Phosphatidylethanolamine, Phosphatidylethanolamine,
C E
P Phosphatidylinositol, Phosphatidylinositol,
1
C16:O
C E
P
ing, as is indicated by the phospholipid phosphorus/mg protein values reported in Table I. The PI and PS values are lower than those reported previously for hepatic microsomes and mitochondria [12]. This may be due to the improved procedure for separating phosphatidylethanolamine (PE), PI and PS. The solvent systems utilized in this study separated the above three phospholipids such that they could be recovered with very little cross-contamination. Earlier solvents utilized [ 131 may not have separated PI and PS sufficiently from PE, which could have resulted in an overestimation of the levels of both PI and PS. Alternatively, the lowered levels of PI and PS may be due to the liquid diets utilized in this study. It is notable that Thompson and Reitz [14] observed (PI + PS) values of 2.9 and 1.7 mol%, respectively, in liver mitochondria
C20:4 C18:2
0.56*0.05 0.81 t-O.05 <0.005 0.591-0.02 0.81 kO.04
3.5 kO.2 3.420.2 n.s. 1.920.3 2.220.2 ns. 2.8 -c 0.6 3.110.3 ns. 18.226.1 IO.52 1.1 ns.
0.56 2 0.02 0.80*0.04
3.720.2 2.6 i: 0.2
from male rats fed liquid diets with or without ethanol. The fatty acid composition of the phospholipid classes in microsomes is shown in Table II. Also reported in Table II is the fatty acid composition of the total phospholipid complement of microsomes. Several statistically significant ethanolrelated alterations in fatty acid composition of microsomal phospholipids were observed and are identified in Table II by asterisks. There were dramatic decreases in the level of palmitic acid in the total microsomal phospholipid complement and the two predominant microsomal phospholipids, phosphatidylcholine (PC) and PE, as a result of ethanol feeding. Palmitic acid levels are also lowered significantly in microsomal PI, as revealed by the paired t test [ll]. There was also an ethanol-induced increase in the level of oleic acid
231
in PE. In addition, the 8,l 1,lCeicosatrienoate was elevated both in the total microsomal phospholipid and in rnicrosomal PI; palmitoleic was decreased significantly in PI due to ethanol feeding. Table111 shows the fatty acid composition of mitochondrial phospholipid classes, both for liquid diet control and ethanol-fed animals. There were even more extensive ethanol-induced changes noted in the mitochondrial phospholipids than in microsomal phosphoglycerides; the statistically significant alterations are identified in Table III by asterisks. Palmitic acid was lowered and oleic acid was elevated due to ethanol feeding in the total phospholipid [3] and in the two predominant mitochondrial phospholipids, PC and PE. Stearic acid was elevated slightly in PC and 8,11,16eicosatrienoate was also elevated in PC. In PC and PE linoleic acid was elevated and arachidonic acid was decreased, both significantly. In contrast linoleic acid levels were decreased appreciably in cardiolipin. Additional ethanol-related alterations were decreased docosahexanoic acid in PI and increased amounts of methyl esters that we were unable to identify in PC, PE and cardiolipin. Several patterns emerged in the ethanol-induced fatty acid alterations which are listed in Table IV. The C18: l/C16:0 ratio was altered in several of the phospholipids of both microsomes and rnitochondria, with there being a shift to higher values in ethanol-fed rats. These phospholipid classes constitute 92% and 90% (mol%) of the total phospholipids in microsomes and mitochondria, respectively. The C20 : 4/C18 : 2 ratio decreased significantly in several of the mitochondrial phospholipids from ethanol-fed rats, but no similar ethanol-induced shifts were observed in microsomal phospholipids. Discussion In this study we have not addressed the effect of the liquid diet itself on rat liver microsomal and mitochondrial phospholipids. In our earlier study [3], however, where the diet and feeding regimen were identical to those in the present investigation, we did measure the effect of the liquid diet on the total phospholipid complement of rat liver mitochondria by analyzing phospholipids from chow-fed animals, The liquid diet alone had no
significant effect on the phospholipid-to-protein ratio. The fatty acid analyses of the total phospholipid complement in all three groups revealed that the liquid diet itself affected the mol percentage of several fatty acids. In every case where a change attributable to the liquid diet was observed, similar alterations were also apparent in the fatty acids from ethanol-fed animals that were equal to or greater than those seen in phospholipids isolated from liquid diet control rats. Those initial observations suggested, but have not proven, that the ethanol-related changes observed in individual phospholipids were a true effect of ethanol on phospholipid metabolism. Another explanation for the differences seen in the present study is that there were shifts in the fatty acid composition attributable to the liquid diet which were prevented by including ethanol in the liquid diet. Our earlier study [3] appears to discount this latter possibility, however. With the exception of the metabolically important phospholipid, PI, being elevated in concentration in liver microsomes of ethanol-fed animals, there were no significant changes in the phospholipid distribution of either the endoplasmic reticulum or mitochondria. These results for mitochondria are in close agreement with those of Thompson and Reitz [14] when their data on males fed a diet containing 34% fat are expressed on a mol% basis. Furthermore, there were no significant changes in the total phospholipid complement of either microsomes or mitochondria, as measured by phospholipid phosphorus/mg organelle protein. Previous measurements of microsomal phospholipid [ 15,161, when expressed on a pmol phospholipid/mg microsomal protein basis, also indicated no significant ethanol-elicited changes in the microsomal phospholipid-to-protein ratio. The phospholipid/protein ratio results obtained for mitochondria are in agreement with earlier reports [ 15,171, including our own [3], on total mitochondrial phospholipid, but contrast slightly with the results of Thompson and Reitz [ 141, who reported an 18% increase in total phospholipid/mg mitochondria protein in male rats fed a diet containing 34% fat. Their high-fat diet was similar to the diet utilized in the present study. An important difference between our studies
232
and the studies of Thompson and Reitz which may influence the magnitude of ethanol-elicited alterations is the duration of ethanol feeding. Their high-fat, male animals were fed the ethanol-containing diet for 46-60 days, whereas all animals in the present investigation were maintained for 31 days on a similar ethanol-containing diet. The increased level of phospholipid in mitochondria for animals fed for longer periods of time is another piece of evidence for the progression of ethanolelicited alterations in liver mitochondria. This phenomenon has been noted earlier in studies of the mitochondrial-associated oxidative phosphorylation system [3,18]. While ethanol feeding, with one exception, elicited no changes in the phospholipid distribution of microsomes and mitochondria, its effect on the fatty acid composition of phospholipids in both organelles was pronounced. For example, Cl6 : 0 was lowered in total microsomal phosmicrosomal PC and PE, total pholipid, mitochondrial phospholipid [3] and mitochondrial PC and PE, with Cl 8 : 1 being elevated in most of the same phospholipids. Our observations on changes in Cl6 : 0 and Cl 8 : 1 in the above phospholipids are in agreement with earlier studies. Thompson and Reitz [14] observed significant ethanol-elicited decreases in Cl6 : 0 in total mitochondrial lipids from male rats fed a high-fat diet; Schilling and Reitz [18] noted similar decreases in inner mitochondrial membranes isolated from the livers of ethanol-fed rats. Moreover, Waring et al. [19] recently observed ethanol-related decreases in Cl6 : 0 in liver mitochondria PC and PE. Likewise, Thompson and Reitz [14] observed an ethanol-elicited increase in the levels of C 18 : 1 in total mitochondrial lipids; Waring et al. [19] also observed a significant increase in this fatty acid in mitochondrial PE. Furthermore, Koivusaari et al. [16] determined that ethanol feeding resulted in an increase in C 18 : 1 in the liver microsome total phospholipid complement. The resulting Cl8 : l/C16 : 0 ratio was increased in all the phospholipids mentioned above, and also in microsomal PI. These observations are consistent with increases in elongation of palmitic acid and desaturation at the A9 position of the resulting stearic acid in liver tissue of ethanol-fed animals.
A shift in the Cl8 : l/C 16 : 0 ratio toward oleic acid could result from higher levels of NADH [20], a co-substrate for the A9 desaturation reaction. This nucleotide is present in higher concentrations, due to a lowered hepatic oxidation-reduction potential which results from increased ethanol oxidation in the livers of alcohol-fed animals [20]. Elevated concentrations of NADH may thus lead to higher levels of phospholipid-associated oleic acid by pushing the A9 desaturation reaction in the direction of product, oleoyl CoA, according to the principle of mass action. It appears unlikely that the increase in the C18: l/C16:0 ratio is related to an increased activity of the A9 desaturase, since a recent study [21] demonstrated that the maximal activity of the A9 desaturase was decreased within 48 h after the rats were placed on an ethanol-containing diet. Since both the feeding protocols and the sex of the animals were different in the two studies, we cannot state unequivocally that the A9 desaturase levels were not elevated in our study, but, in the literature, evidence is lacking which would suggest an increase in the activity of this enzyme. The fatty acid compositions of mitochondrial phospholipids are altered more than are the same phospholipid classes in microsomes. For example, the two major mitochondrial phospholipids, PC and PE, have elevated C 18 : 2 levels and decreased C20 : 4 concentrations in preparations from ethanol-fed rats. These results on the individual phospholipids are in agreement with earlier studies which provided evidence for a lowered C20 : 4/C18 : 2 ratio in total mitochondrial phospholipids [ 15,22,23]. The C20 : 3 is also elevated in PC, as it is in the total microsomal complement and microsomal PI. These observations are consistent with the possibility that there is an ethanol-related decrease in the synthesis of arachidonic acid from linoleic acid. The hepatic desaturation elongation system, which includes the A6 and A5 desaturases, may be decreased as a result of chronic ethanol feeding. Nervi et al. [21] have determined that the activities of the A6 and A5 desaturases are lowered in female rats after 48 h of ethanol administration, which demonstrates the potential of ethanol, when consumed, to decrease activities involved in arachidonic acid synthesis. Since the mode and duration of ethanol adminis-
233
tration and animal sex were different in their and our studies, it will be necessary to measure the response of the desaturases to our feeding protocol (see Methods) in order to establish the relationship between these enzymes and the alterations in fatty acid patterns reported in this paper. The present study has emphasized the importance of systematically investigating the effect of chronic alcoholism on the desaturase system. Linoleic acid levels decrease in cardiolipin, rather than increase as is the case with mitochondrial PC and PE; this observation is in agreement with the analyses of Waring et al. [19] on the fatty acid composition of liver mitochondrial cardiolipin. The contrasting effects of ethanol on linoleic acid levels in cardiolipin as compared to the other mitochondrial phospholipids probably relates to the fact that cardiolipin is synthesized in the mitochondrion [24] whereas the other phospholipids are microsomally derived. The alterations in the fatty acid composition in cardiolipin, as well as in mitochondrial PC and PE, emphasize the potential of individual phospholipids, serving as boundary lipids, to modulate the activities of enzymes with which they may associate in the mitochondrial membrane. Acknowledgments
This work was supported by NIAAA grant 02887 and a grant from the North Carolina Alcoholism Research Authority. C.C.C. is the recipient of a NIAAA Research Development Award AAOOo43. References 1 Gordon, E. (1973) J. Biol. Chem. 248, 8271-8280 2 Cederbaum, A., Lieber, C. and Rubin, E. (1974) Biochem. Biophys. 165, 560-569
Arch.
3 Spach, P., Parce, J. and Cunningham, 4 5 6 7 8 9 10 11
C. (1979) B&hem.
J.
178, 23-33 McPhail, L. and Cunningham, C. (1975) Biochemistry 14, 1122-1131 Cunningham, C. and George, D. (1975) J. Biol. Chem. 250, 2036-2044 Rottenburg, H., Robertson, D. and Rubin, E. (1980) Lab. Invest. 42, 318-326 Hosein, E., Hofmann, I. and Linder, E. (1977) Arch. Biothem. Biophys. 183, 64-72 DeCarli, L. and Lieber, C. (1967) J. Nutr. 91, 331-336 Bligh, E. and Dyer, W. (1959) Can. J. B&hem. Physiol. 37, 91 I-917 Chalvardjian, A. and Rudnicki, E. (1970) Anal. B&hem. 36, 225-226 Engelman, L., Frane, J. and Jemuich, J. (1977) in BMDP-77 Biomedical Computer Programs P-Series (Dixon, W. and Brown, M., eds.), pp. 170- 184, University of California, Press, Berkeley
12 White, D. (1973) in Form and Function of Phospholipids (Ansell, G., Hawthorne, J. and Dawson, R., eds.), pp. 441-482, Elsevier Scientific Publishing Company, Amsterdam 13 Colbeau, A., Nachbaur, J. and Vignais, P. (1971) Biochim. Biophys. Acta 249, 462-492 14 Thompson, J. and Reitz, R. (1978) Lipids 13, 540-550 15 French, S., Ihrig, T. and Morin, R. (1970) Q. J. Stud. Ale. 31, 801-809 16 Koivusaari, U., Norling, A., Lang, M. and Heitanen, E. (1981) Toxicology 20, 173-183 17 Lundquist, C., Kiessling, K. and Pilstrom, L. (1966) Acta Chem. Stand. 20, 275 l-2754 18 Schilling, R. and Reitz, R. (1980) B&him. Biophys. Acta 603, 266-277 19 Waring, A., Rottenburg, H., Ohnishi, T. and Rubin, E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2582-2586 20 Christiansen, E. and Higgins, J. (1979) in Biochemistry and Pharmacology of Ethanol (Majchrowicz, E. and Noble, E., eds.), Vol. 1, pp. 191-247, Plenum Press, New York 21 Nervi, A., Peluffo, R., Brenner, R. and Leikin, A. (1980) Lipids 15, 263-268 22 French, S., Ihrig, T., Shaw, G., Tanaka, T. and Norum, M. (1971) Res. Commun. Chem. Pathol. Pharmacol. 2,567-585 23 Thompson, J. and Reitz, R. (1976) Ann. N.Y. Acad. Sci. 273, 194-204 24 Thompson, G. (1980) The Regulation of Membrane Lipid Metabolism, pp. 84-104, CRC Press, Boca Raton, FL
225
BBA 5 1142
EFFECT OF ETHANOL CONSUMPTION ON THE PHOSPHOLIPID LIVER MICROSOMES AND MITOCHONDRIA CAROL
C. CUNNINGHAM,
STEPHEN
FILUS,
RALPH
E. BOITENUS
and PRISCILLA
COMPOSITION
OF RAT
I. SPACH
Department of Biochemistry Bowman Gray School of Medicine, Wake Forest Unioersity, Winston-Salem, NC 27103 (U.S.A.) (Received
January
26th, 1982)
Key words: Ethanol; Microsome; Mitochondria; Phospholipid; (Rat liver)
Male Sprague-Dawley rats were maintained for 31 days on a liquid diet containing 36% of calories as ethanol. Pair-fed controls were administered a similar diet, but with maltose-dextrin isocalorically substituted for ethanol. A phospholipid analysis has been carried out in liver microsomes and mitochondria isolated from the two groups of animals. The phospholipid phosphorus/protein ratio was not significantly different in the organelles of the ethanol-fed animals as compared to the same organelles of liquid diet controls, which indicates that ethanol feeding did not influence the total phospholipid content of microsomes and mitochondria. The phospholipid distribution within organelles was not changed, except for a significant increase in the phosphatidylinositol content of microsomes from ethanol-fed animals. The fatty acid compositions of both microsomal and mitochondrial phospholipids were significantly altered by ethanol feeding. In microsomes from ethanol-fed rats, palmitic acid levels were lowered in the total phospholipid fraction, phosphatidylcholine and phosphatidylethanolamine; oleic acid levels were elevated in microsomal phosphatidylethanolamine. In mitochondria from ethanol-fed animals, palmitic and arachidonic acid were lowered in phosphatidylcholine and phosphatidylethanolamine. Oleic and linoleic acid were elevated in the same phospholipids. In contrast, IhroIeic acid levels in cardiolipin were depressed significantly. These alterations in the fatty acid composition are suggestive of ethanol-induced changes in fatty acid desaturation activities.
Introduction
[6,7] activities may be linked, at least in part, to ethanol-induced changes in membrane phospholipids. It would appear, however, that changes observed in those activities associated with the oxidative phosphorylation system cannot be linked with changes in the fatty acid composition of bulk-phase phospholipids from mitochondrial membranes. In an earlier study [3] we compared the energy-linked properties and associated enzyme activities, and the fatty acid composition of the total phospholipids in mitochondria from rats fed either chow or a control liquid diet, or an ethanol-containing liquid diet. Few differences between the fatty acid compositions of liver mitochondria phospholipids from liquid diet controls and liquid diet ethanol-fed
Several studies have demonstrated that the oxidative phosphorylation system in rat liver mitochondria is adversely affected by chronic ethanol consumption [l-3]. The oxidative phosphorylation system, being membrane-associated, requires phospholipids for its proper functioning. Moreover, those enzymes comprising the oxidative phosphorylation system require phospholipids for expression of their activities [4,5]. Recent studies suggest that changes related to ethanol feeding in both the mitochondrial respiratory [6] and ATPase Abbreviations: PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine. OOOS-2760/82/0000-0000/$02.75
0 1982 Elsevier Biomedical
Press
226
animals were seen, whereas several significant changes in energy-linked functions and associated enzyme activities were noted. In contrast, when chow-fed and liquid diet control animals were compared, dramatic differences in the fatty acid composition of mitochondrial phospholipids were noted, but no differences were observed in either energy-linked functions or associated enzyme activities. The above observations tend to discount that changes in the fatty acid composition of bulk phase lipids are responsible for ethanol-induced alterations in mitochondrial associated energy-linked functions. They did not eliminate the possibility, however, that alterations in either the concentration or the fatty acid composition of a particular phospholipid could be responsible for some of the ethanol-induced alterations in the oxidative phosphorylation system. In this communication we report the concentrations of mitochondrial phospholipid classes isolated from the livers of ethanol-fed rats. In addition, the fatty acid composition of each mitochondrial phospholipid was measured, with the ethanol-induced changes being reported in this paper. The changes in fatty acid composition demonstrate the possibility that alterations in individual phospholipids could result in changes in the activity of membrane-associated enzymes, since a particular phospholipid, rather than the total phospholipid complement, might serve as a boundary lipid for an enzyme whose activity was altered by ethanol feeding. In addition to measuring the ethanol-induced alterations in mitochondrial phospholipid composition, we have also determined those changes in phospholipids which occur in microsomes. Since the endoplasmic reticulum is the site of synthesis for most mitochondrial phospholipids, a comparison of the ethanol-induced alterations in phospholipids from both organelles provides us with information that suggests the origin of the changes in the mitochondrial phospholipids. Experimental procedures Materials Sprague-Dawley rats were obtained from Charles River Breeding Laboratories, North Wilmington, MA. Lieber/DeCarli liquid diets were
obtained from Bio-Serv, Frenchtown, NJ. Phospholipid standards were purchased from Serdary Research Laboratories, London, Ontario, Canada. Other reagents, including supplies for fatty acid analyses, were obtained as listed in Spach et al. [3]. Methods Diets, and preparation of mitochondria and microsomes. Male Sprague-Dawley rats weighing 150-250 g were fed for 31 days on a nutritionally adequate liquid diet [8] in which ethanol provided 36% of the total calories. Pair-fed control rats received the same diet, but with maltose-dextrin isocalorically substituted for ethanol. Preparation of tightly coupled mitochondria and protein determinations were carried out as described previously [3]. Microsomes were prepared from the postmitochondrial (8700 X g) supernatant by the following procedure. The supernatant was centrifuged at 17300 X g for 10 min to remove lysosomes and then at 78500 X g for 90 min to sediment the microsomes. The microsomes were resuspended in 40 ml of 0.25 M sucrose, pH 7.4, and recentrifuged at 78 500 X g for 90 min. The washed microsomes were resuspended in 10 ml of 0.25 M sucrose, pH 7.4. Initial experiments measuring marker enzymes demonstrated that the microsomes were minimally contaminated with other cell components. Values obtained for NADPHcytochrome c reductase activity indicate that the microsomal fraction is enriched by as much as 22-fold when compared to mitochondrial preparations (microsomes, 67 nmol/min per mg protein; mitochondria, 3 nmol/min per mg protein). Phospholipid analyses of mitochondria and microsomes. Lipids were extracted from cell organelles immediately after their preparation by the procedure of Bligh and Dyer [9] to minimize hydrolysis by phospholipases, and aliquots were taken for lipid phosphorus analyses [IO]. Either 30 mg mitochondrial protein or 16 mg microsomal protein were extracted to obtain the lipid complement. Phospholipids within the extract were fractionated by two-dimensional thin-layer chromatography on silica gel H. The chromatography plate was developed in chloroform/acetone,/ methanol/acetic acid/water (10 : 4 : 2 : 3 : 1, V/V) and stored overnight in a nitrogen atmosphere.
227
ages rt S.E. for the numbers indicated. Statistical analyses were performed using the t test for two means [ 111.
The plate was then developed in a second dimension with butanol/acetic acid/water (80: 26 : 26, v/v). This combination of solvents provided good separation of all phospholipid classes, including phosphatidylserine (PS) and phosphatidylinositol (PI). To locate the phospholipids, the chromatogram was sprayed lightly with dichlorofluorescein in 95% ethanol and then illuminated with an ultraviolet lamp. The phospholipid spots, identified on the chromatogram by use of purified standards, were scraped from the plate. The phospholipids were eluted from silicic acid by repeated washing with chloroform/methanol/water (1: 2 : 0.2, v/v). This extraction procedure is quantitative for all phospholipids except sphingomyelin; the recovery is 80-85% with the latter (King, L. and Waite, M., personal communication). The phospholipid extracts were analyzed for lipid phosphorus levels and for fatty acid composition as outlined previously [3]. The fatty acids were identified by comparing their elution times with those of purified standards utilizing a Varian 3700 gas chromatograph. Quantitation was carried out with a Varian CDS-1 11 data processor. The values reported in the tables are the averTABLE
Results
The livers from animals fed the ethanol-containing diets demonstrated fatty liver, as assessed by their increased size and their excess lipid. Table I demonstrates that the phospholipid distribution in microsomes and ~t~hondria isolated from these livers was minimally affected by ethanol feeding. Of all the phospholipids in both organelles, only microsomal PI was altered as a resuit of the ethanol diet. This metabolically important phospholipid was increased 66% in microsomes, but its level in mitochondria was not affected in the ethanol-fed animals. Cardiolipin was lowered in liver microsomes from ethanol-fed rats, which suggested that ethanol microsomes were less contaminated with mit~hondria since c~diolipin is felt to be localized to the latter organelle. The phospholipid-toprotein ratio was not altered in either liver microsomes or mitochondria as a result of ethanol feed-
I
PHOSPHOLIPID CALLY
DISTRIBUTION
IN LIVER
MICROSOMES
AND
MITOCHONDRIA
OF RATS FED ETHANOL
CHRONI-
For microsomes, n = 8 control and 9 ethanol preparations. Total phospholipid: control, 441 -t 24, nmol phospholipid phosphorus/mg protein; ethanol, 481.227 nmol phospholipid phosphorus/mg protein. For mitochondria, n = 11 control preparations and 7 ethanol preparations. Total phospholipid: control, 184% 10 nmol phospholipid phosphorus/mg protein; ethanol, 19Ok9 nmol phospholipid phospho~s/mg protein. Ail values are mean* SE. Phospholipid
type
Distribution
(mol’k;)
Microsomes
Phosphatidybholine Lysophosphatidylcholine Phosphatidylethanolamine Lysophosphatidylethanolamine Phosphatidylinositol Phosphatidyise~ne Sphingomyehn ’ Cardiolipin ’ Significantly
different different
Control
Ethanol
Control
Ethanol
66.0 k1.5 1.01*0.27 23.1 =I.4 0.7520.22 3.3620.64 2.38 2 0.68 2.6lt0.30 0.797tO.17
64.4 -t 1.8 1.36f0.23 23.0 2 1.2 1.18-‘0.46 5.58*0.51 a I .94 * 0.27 2.19-0.33 0.33 5 0.09 =
48.8 12.1 o&=0.19 38.8 - 1.7 0.44*0.16 1.89kO.31 0.67f0.16 0.63-0.16 8.2 kO.90
53.0 * 1.7 0.54~0.16 34.6 * 1.8 0.42-tO.18 2.2920.30 0.98 kO.26 0.63CO.15 7.54+0.71
from control (P ~0.025). may be as much as 20% too low due to difficulties from control (P ~0.05).
b The values for sphingomyel~n f Significantly
Mit~hondria
with its extraction
from silicic acid.
TABLE
II
FATTY
ACID
DISTRIBUTION
The data were obtained Phosphohpid
FOR MICROSOMAL
from microsomal
type
PHOSPHOLIPIDS
preparations
isolated
from nine pairs of pair-fed
Animal
Fatty
acid distribution
animals.
Values are meant-
S.E.
(mol%)
group 16:O
Phosphatidylchohne
16:l
18:O
2.2-cO.4 1.8i0.2
23.2kO.7 24.5 -c 0.8
Control Ethanol-fed
22.5 to.9 16.9-0.6
Lysophosphatidylcholine
Control Ethanol-fed
28.8‘4.0 29.1-t 1.8
4.7= 1.2 4.420.3
28.9* 3.5 30.5 * 1.7
Phosphatidylethanolamine
Control Ethanol-fed
21.9’-2.1 16.1) 1.1 *
1.4kO.2 1.110.3
31.4t3.0 27.8r2.0
Lysophosphatidylethanolamine
Control Ethanol-fed
25.31 1.8 26.6 rt 2.2
5.4* 1.0 7.02 I.0
23.71-2.8 24.9k2.3
Phosphatidylinositol
Control
***
9.62 1.4 6.5fl.l *
Ethanol-fed
2.220.5 I.0f0.2
44.5 * 4.0 45.2-c 1.8
+
Phosphatidylserine
Control Ethanol-fed
16.2’1.9 15.2-c 1.7
3.5 -CO.6 3.950.8
38.1 “3.6 35.7k3.6
Sphingomyelin
Control Ethanol-fed
30.5 * 3.0 30.422.8
4.0-t0.8 5.350.9
13.92 1.7 16.6~ 1.8
Total phosphohpid
Control Ethanol-fed
17.5’00.6 13.8kO.4
1.2’0.4 0.6iO.l
23.3* 1.0 22.6 -c 2.6
a The predominant fatty acids were 22: 5 (control, * P (0.05; **p co.01; ***p
III
FATTY
ACID
DISTRIBUTION
The numbers in parentheses *** P ~0.001. Phosphohpid
6.1 mol%; ethanol,
FOR MITOCHONDRIAL
indicate
the number
8.5 mol%) and 24: 0 (control,
5.1 mol%; ethanol,
3.6 mol%).
PHOSPHOLIPIDS
of pairs of animals
Animal
type
***
used for analyses.
Fatty
Values are meani
acid distribution
S.E. * P (0.05;
** P
(mol%)
group 16:0
Phosphatidylcholine
Control Ethanol-fed
(20)
19.9kO.6 16.6kO.6
16:
***
I
18:O
1.9kO.2 1.9*0.1
20.4-tO.6 23.1 50.8
5.5% 1.5 7.2k2.3
14.3k2.1 17.0 ‘- 3.0
1.3r0.2 1.6*0.2
26.5~ 25.9i
Control Ethanol-fed
31.9k4.5 27.41- 2.5
Control Ethanol-fed
17.9*0.8 15.5*0.9
Control Ethanol-fed
30.9k4.8 32.3k4.3
6.5 kO.9 6.1 ‘2.3
16.8& 1.9 13.0*4.0
Phosphatidylinositol(20)
Control Ethanol-fed
16.0+2.0 17.6 2 2.4
4.4kO.8 5.3% 1.3
30.1 k2.3 30.9* 3.2
Phosphatidylserine
Control Ethanol-fed
26.1* 2.6 20.4k2.8
4.2kO.6 3.2’1.0
23.2k2.9 21.2’4.1
Control Ethanol-fed
7.1 eo.7 8.5-‘1.1
2.9kO.4 2.41-0.3
Lysophosphatidylchohne Phosphatidylethanolamine
Lysophosphatidylethanolamine
Cardiohpin
(17)
(11)
(8)
(19) (8)
*
1.2 1.9
2.2kO.3 3.41-0.6
**
229
18:l
18:2
20:3
20:4
2216
20:5, 22:4 2215, 24:0,
Unidentified
24:l 11.4*0.7
1.1*0.1
2.11-0.4 2.OkO.2
2.920.4
2.120.6
19.9k2.8 22.9-c 1.3
1.2-t0.3
10.8kO.5
1.5kO.2
4.2-t0.8
4.1 -to.9 3.6-cO.6
1.1 eo.3 1.320.5
3.4* 1.2 2.6kO.7
3.4-0.7 2.O-tO.6
5.622.5 5.82 1.2
7.0* 3.6 6.1k2.2
5.5 kO.6 6.5kO.3
0.4kO.1 1.82 1.0
4.62 1.0 4.3eo.5
3.1 to.7 2.3-t0.5
3.820.5 6.4* 1.8
19.2% 1.8 15.Ok2.3
4.7% 1.5 4.2-t 1.1
2.121.6 2.1k1.2
3.8kO.9 2.OeO.7
6.4* 1.5 7.6* 1.9
7.722.1 4.7* 1.3
7.1’-1.0 7.5eo.4
6.024.1 2.5 eO.3
0.7kO.2 3.o-co.9
22.1e3.8 25.1 c2.5
1.5-0.4 1.9*0.5
3.3f0.9 4.02 1.0
2.3% 1.0 3.2kO.8
12.3kl.6 11.0r1.3
3.3 kO.7 3.2% 1.1
1.4-0.9 0.9co.4
13.4k3.1 12.6k2.1
3.5kO.7 3.720.5
4.6-f 1.0 5.121.3
3.6” 1.2 8.7~3.2
14.3k2.5 12.4* 1.8
3.1 eo.7 4.OkO.3
I.32 1.0 0.6f0.3
1.3 1.5
4.1%1.1 3.020.9
Il.35 1.8 a 12.8k3.4
14.2k3.9 10.9” 1.6
9.6eo.7 11.0*0.4
8.1 e-o.5 8.5 eO.4
0.6-tO.2 1.4kO.l
27.6* 1.5 28.6kO.5
5.2kO.3 4.5kO.6
13.3kO.7 13.5*0.4 13.1k1.2 14.3-c 1.5 11.0*0.6 13.OkO.7
*
18:l
11.0*0.3 13.1kO.6
18:2
**
15.Ok2.4 18.Ok2.5
8.4kO.3 9.820.4
17.Ok3.6 20.622.4 1.8t- 1.0 4.2t-2.1
*
3.3a 4.oc
**
20:3
**
0.620.1 1.5kO.l 0.7-to.3 0.420.4
10.1 k2.2 4.9-cO.8
0.4kO.1 0.8 r0.2
20:4
***
22~6
29.6’0.9 25.2kl.l
**
3.5* 1.8 4.3*2.2
3.3eO.2 4.1kO.2
4.OkO.8 3.1t1.1
4.2-t 1.1 10.723.9
7.6* 1.1 12.Ok3.6
6.6kO.6 5.8 20.6
2.5k1.3 3.5e1.1
3.7kO.3 5.810.7
6.8-1.9 14.7k6.2
7.1k2.1 14.2k4.8
14.62 1.8 10.4% 1.1
5.7a2.5 2.3-cO.9
1.420.6 0.9eo.5
1.6kO.7 3.9k3.0
7.723.8 2.3k1.1
11.3-cO.8 11.8-1.1
2.8eO.5 3.1 kO.5
1.3kO.3 1.2~0.4
18.4k2.0 14.3k2.1
3.9t0.8 2.0-t0.4
16.822.8 10.3* 1.4
3.3-cO.6 4.4* 2.2
1.1*0.3 3.7e3.5
5.9-2.1 4.8* 1.8
2.020.2 5.2k2.3
2.420.3 1.8kO.S
51.3k2.0 42.8k3.9
*
1.3 1.7 *
Unidentified
1.5eO.2 1.7kO.3
4.120.2 5.950.8
24.OkO.8 23.2) 1.4
26.72 21.9*
20:5, 22~4, 22:5, 24:0 24: 1
4.2-cO.9 6.7*2.7
3.4*0.3 3.OkO.3
10.3kO.4 13.3% 1.3 *
*
2.6-0.7 2.3f0.6
6.OkO.8 5.6kl.O
5.OkO.7 8.0* 1.7
4.822.1 2.3-cO.7
7.9* 1.5 17.6k7.5
6.7* 1.3 11.9k3.5
1.7-co.3 1.3*0.2
1.8kO.3 4.0 * 2.0
*
**
**
4.6kO.6 7.4* I.2 *
230 TABLE
IV
ETHANOL-INDUCED PHOSPHOLIPIDS
ALTERATIONS
IN PARAMETERS
RELATED
TO FATTY
ACID
COMPOSITION
OF MEMBRANE
The data on microsomal lipids were obtained from nine pairs of pair-fed animals. The data for mitochondrial phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol were obtained from 20, 19 and 19 pairs of pair-fed animals respectively. The values were obtained by first dividing the mol% value for oleic acid by that of palmitic acid and then calculating the average-t S.E. The C20:4/C18:2 ratio was obtained in like manner. C, phospholipid from control animals; E, phospholipid from ethanol-fed animals.
Organelle
Phospholipid
Microsomes
Total, C Total, E
C18:
P Phosphatidylcholine, Phosphatidylcholine,
C E
P Phosphatidylethanolamine, Phosphatidylethanolamine,
C E
P Phosphatidylinositol, Phosphatidylinositol,
C E
P Mitochondria
Phosphatidylcholine, Phosphatidylcholine,
C E
P Phosphatidylethanolamine, Phosphatidylethanolamine,
C E
P Phosphatidylinositol, Phosphatidylinositol,
1
C16:O
C E
P
ing, as is indicated by the phospholipid phosphorus/mg protein values reported in Table I. The PI and PS values are lower than those reported previously for hepatic microsomes and mitochondria [12]. This may be due to the improved procedure for separating phosphatidylethanolamine (PE), PI and PS. The solvent systems utilized in this study separated the above three phospholipids such that they could be recovered with very little cross-contamination. Earlier solvents utilized [ 131 may not have separated PI and PS sufficiently from PE, which could have resulted in an overestimation of the levels of both PI and PS. Alternatively, the lowered levels of PI and PS may be due to the liquid diets utilized in this study. It is notable that Thompson and Reitz [14] observed (PI + PS) values of 2.9 and 1.7 mol%, respectively, in liver mitochondria
C20:4 C18:2
0.56*0.05 0.81 t-O.05 <0.005 0.591-0.02 0.81 kO.04
3.5 kO.2 3.420.2 n.s. 1.920.3 2.220.2 ns. 2.8 -c 0.6 3.110.3 ns. 18.226.1 IO.52 1.1 ns.
0.56 2 0.02 0.80*0.04
3.720.2 2.6 i: 0.2
from male rats fed liquid diets with or without ethanol. The fatty acid composition of the phospholipid classes in microsomes is shown in Table II. Also reported in Table II is the fatty acid composition of the total phospholipid complement of microsomes. Several statistically significant ethanolrelated alterations in fatty acid composition of microsomal phospholipids were observed and are identified in Table II by asterisks. There were dramatic decreases in the level of palmitic acid in the total microsomal phospholipid complement and the two predominant microsomal phospholipids, phosphatidylcholine (PC) and PE, as a result of ethanol feeding. Palmitic acid levels are also lowered significantly in microsomal PI, as revealed by the paired t test [ll]. There was also an ethanol-induced increase in the level of oleic acid
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in PE. In addition, the 8,l 1,lCeicosatrienoate was elevated both in the total microsomal phospholipid and in rnicrosomal PI; palmitoleic was decreased significantly in PI due to ethanol feeding. Table111 shows the fatty acid composition of mitochondrial phospholipid classes, both for liquid diet control and ethanol-fed animals. There were even more extensive ethanol-induced changes noted in the mitochondrial phospholipids than in microsomal phosphoglycerides; the statistically significant alterations are identified in Table III by asterisks. Palmitic acid was lowered and oleic acid was elevated due to ethanol feeding in the total phospholipid [3] and in the two predominant mitochondrial phospholipids, PC and PE. Stearic acid was elevated slightly in PC and 8,11,16eicosatrienoate was also elevated in PC. In PC and PE linoleic acid was elevated and arachidonic acid was decreased, both significantly. In contrast linoleic acid levels were decreased appreciably in cardiolipin. Additional ethanol-related alterations were decreased docosahexanoic acid in PI and increased amounts of methyl esters that we were unable to identify in PC, PE and cardiolipin. Several patterns emerged in the ethanol-induced fatty acid alterations which are listed in Table IV. The C18: l/C16:0 ratio was altered in several of the phospholipids of both microsomes and rnitochondria, with there being a shift to higher values in ethanol-fed rats. These phospholipid classes constitute 92% and 90% (mol%) of the total phospholipids in microsomes and mitochondria, respectively. The C20 : 4/C18 : 2 ratio decreased significantly in several of the mitochondrial phospholipids from ethanol-fed rats, but no similar ethanol-induced shifts were observed in microsomal phospholipids. Discussion In this study we have not addressed the effect of the liquid diet itself on rat liver microsomal and mitochondrial phospholipids. In our earlier study [3], however, where the diet and feeding regimen were identical to those in the present investigation, we did measure the effect of the liquid diet on the total phospholipid complement of rat liver mitochondria by analyzing phospholipids from chow-fed animals, The liquid diet alone had no
significant effect on the phospholipid-to-protein ratio. The fatty acid analyses of the total phospholipid complement in all three groups revealed that the liquid diet itself affected the mol percentage of several fatty acids. In every case where a change attributable to the liquid diet was observed, similar alterations were also apparent in the fatty acids from ethanol-fed animals that were equal to or greater than those seen in phospholipids isolated from liquid diet control rats. Those initial observations suggested, but have not proven, that the ethanol-related changes observed in individual phospholipids were a true effect of ethanol on phospholipid metabolism. Another explanation for the differences seen in the present study is that there were shifts in the fatty acid composition attributable to the liquid diet which were prevented by including ethanol in the liquid diet. Our earlier study [3] appears to discount this latter possibility, however. With the exception of the metabolically important phospholipid, PI, being elevated in concentration in liver microsomes of ethanol-fed animals, there were no significant changes in the phospholipid distribution of either the endoplasmic reticulum or mitochondria. These results for mitochondria are in close agreement with those of Thompson and Reitz [14] when their data on males fed a diet containing 34% fat are expressed on a mol% basis. Furthermore, there were no significant changes in the total phospholipid complement of either microsomes or mitochondria, as measured by phospholipid phosphorus/mg organelle protein. Previous measurements of microsomal phospholipid [ 15,161, when expressed on a pmol phospholipid/mg microsomal protein basis, also indicated no significant ethanol-elicited changes in the microsomal phospholipid-to-protein ratio. The phospholipid/protein ratio results obtained for mitochondria are in agreement with earlier reports [ 15,171, including our own [3], on total mitochondrial phospholipid, but contrast slightly with the results of Thompson and Reitz [ 141, who reported an 18% increase in total phospholipid/mg mitochondria protein in male rats fed a diet containing 34% fat. Their high-fat diet was similar to the diet utilized in the present study. An important difference between our studies
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and the studies of Thompson and Reitz which may influence the magnitude of ethanol-elicited alterations is the duration of ethanol feeding. Their high-fat, male animals were fed the ethanol-containing diet for 46-60 days, whereas all animals in the present investigation were maintained for 31 days on a similar ethanol-containing diet. The increased level of phospholipid in mitochondria for animals fed for longer periods of time is another piece of evidence for the progression of ethanolelicited alterations in liver mitochondria. This phenomenon has been noted earlier in studies of the mitochondrial-associated oxidative phosphorylation system [3,18]. While ethanol feeding, with one exception, elicited no changes in the phospholipid distribution of microsomes and mitochondria, its effect on the fatty acid composition of phospholipids in both organelles was pronounced. For example, Cl6 : 0 was lowered in total microsomal phosmicrosomal PC and PE, total pholipid, mitochondrial phospholipid [3] and mitochondrial PC and PE, with Cl 8 : 1 being elevated in most of the same phospholipids. Our observations on changes in Cl6 : 0 and Cl 8 : 1 in the above phospholipids are in agreement with earlier studies. Thompson and Reitz [14] observed significant ethanol-elicited decreases in Cl6 : 0 in total mitochondrial lipids from male rats fed a high-fat diet; Schilling and Reitz [18] noted similar decreases in inner mitochondrial membranes isolated from the livers of ethanol-fed rats. Moreover, Waring et al. [19] recently observed ethanol-related decreases in Cl6 : 0 in liver mitochondria PC and PE. Likewise, Thompson and Reitz [14] observed an ethanol-elicited increase in the levels of C 18 : 1 in total mitochondrial lipids; Waring et al. [19] also observed a significant increase in this fatty acid in mitochondrial PE. Furthermore, Koivusaari et al. [16] determined that ethanol feeding resulted in an increase in C 18 : 1 in the liver microsome total phospholipid complement. The resulting Cl8 : l/C16 : 0 ratio was increased in all the phospholipids mentioned above, and also in microsomal PI. These observations are consistent with increases in elongation of palmitic acid and desaturation at the A9 position of the resulting stearic acid in liver tissue of ethanol-fed animals.
A shift in the Cl8 : l/C 16 : 0 ratio toward oleic acid could result from higher levels of NADH [20], a co-substrate for the A9 desaturation reaction. This nucleotide is present in higher concentrations, due to a lowered hepatic oxidation-reduction potential which results from increased ethanol oxidation in the livers of alcohol-fed animals [20]. Elevated concentrations of NADH may thus lead to higher levels of phospholipid-associated oleic acid by pushing the A9 desaturation reaction in the direction of product, oleoyl CoA, according to the principle of mass action. It appears unlikely that the increase in the C18: l/C16:0 ratio is related to an increased activity of the A9 desaturase, since a recent study [21] demonstrated that the maximal activity of the A9 desaturase was decreased within 48 h after the rats were placed on an ethanol-containing diet. Since both the feeding protocols and the sex of the animals were different in the two studies, we cannot state unequivocally that the A9 desaturase levels were not elevated in our study, but, in the literature, evidence is lacking which would suggest an increase in the activity of this enzyme. The fatty acid compositions of mitochondrial phospholipids are altered more than are the same phospholipid classes in microsomes. For example, the two major mitochondrial phospholipids, PC and PE, have elevated C 18 : 2 levels and decreased C20 : 4 concentrations in preparations from ethanol-fed rats. These results on the individual phospholipids are in agreement with earlier studies which provided evidence for a lowered C20 : 4/C18 : 2 ratio in total mitochondrial phospholipids [ 15,22,23]. The C20 : 3 is also elevated in PC, as it is in the total microsomal complement and microsomal PI. These observations are consistent with the possibility that there is an ethanol-related decrease in the synthesis of arachidonic acid from linoleic acid. The hepatic desaturation elongation system, which includes the A6 and A5 desaturases, may be decreased as a result of chronic ethanol feeding. Nervi et al. [21] have determined that the activities of the A6 and A5 desaturases are lowered in female rats after 48 h of ethanol administration, which demonstrates the potential of ethanol, when consumed, to decrease activities involved in arachidonic acid synthesis. Since the mode and duration of ethanol adminis-
233
tration and animal sex were different in their and our studies, it will be necessary to measure the response of the desaturases to our feeding protocol (see Methods) in order to establish the relationship between these enzymes and the alterations in fatty acid patterns reported in this paper. The present study has emphasized the importance of systematically investigating the effect of chronic alcoholism on the desaturase system. Linoleic acid levels decrease in cardiolipin, rather than increase as is the case with mitochondrial PC and PE; this observation is in agreement with the analyses of Waring et al. [19] on the fatty acid composition of liver mitochondrial cardiolipin. The contrasting effects of ethanol on linoleic acid levels in cardiolipin as compared to the other mitochondrial phospholipids probably relates to the fact that cardiolipin is synthesized in the mitochondrion [24] whereas the other phospholipids are microsomally derived. The alterations in the fatty acid composition in cardiolipin, as well as in mitochondrial PC and PE, emphasize the potential of individual phospholipids, serving as boundary lipids, to modulate the activities of enzymes with which they may associate in the mitochondrial membrane. Acknowledgments
This work was supported by NIAAA grant 02887 and a grant from the North Carolina Alcoholism Research Authority. C.C.C. is the recipient of a NIAAA Research Development Award AAOOo43. References 1 Gordon, E. (1973) J. Biol. Chem. 248, 8271-8280 2 Cederbaum, A., Lieber, C. and Rubin, E. (1974) Biochem. Biophys. 165, 560-569
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12 White, D. (1973) in Form and Function of Phospholipids (Ansell, G., Hawthorne, J. and Dawson, R., eds.), pp. 441-482, Elsevier Scientific Publishing Company, Amsterdam 13 Colbeau, A., Nachbaur, J. and Vignais, P. (1971) Biochim. Biophys. Acta 249, 462-492 14 Thompson, J. and Reitz, R. (1978) Lipids 13, 540-550 15 French, S., Ihrig, T. and Morin, R. (1970) Q. J. Stud. Ale. 31, 801-809 16 Koivusaari, U., Norling, A., Lang, M. and Heitanen, E. (1981) Toxicology 20, 173-183 17 Lundquist, C., Kiessling, K. and Pilstrom, L. (1966) Acta Chem. Stand. 20, 275 l-2754 18 Schilling, R. and Reitz, R. (1980) B&him. Biophys. Acta 603, 266-277 19 Waring, A., Rottenburg, H., Ohnishi, T. and Rubin, E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2582-2586 20 Christiansen, E. and Higgins, J. (1979) in Biochemistry and Pharmacology of Ethanol (Majchrowicz, E. and Noble, E., eds.), Vol. 1, pp. 191-247, Plenum Press, New York 21 Nervi, A., Peluffo, R., Brenner, R. and Leikin, A. (1980) Lipids 15, 263-268 22 French, S., Ihrig, T., Shaw, G., Tanaka, T. and Norum, M. (1971) Res. Commun. Chem. Pathol. Pharmacol. 2,567-585 23 Thompson, J. and Reitz, R. (1976) Ann. N.Y. Acad. Sci. 273, 194-204 24 Thompson, G. (1980) The Regulation of Membrane Lipid Metabolism, pp. 84-104, CRC Press, Boca Raton, FL