Studies on the composition and structure of the phosphatidylcholine, phosphatidylethanolamine and triglyceride isolated from rabbit liver

BIOCHIMICA

ET BIOPHYSICA

ACTA

137

nnA 5.5073

STUDIES ON THE COMPOSITION AND STRUCTURE OF THE PHOSPHATIDYLCHOLINE, PHOSPHATIDYLETHANOLAMINE AND TRIGLYCERIDE ISOLATED FROM RABBIT LIVER

J. H. MOORE Naiional

AdD

D. L. WILLIAMS

Institule for

(Received

Research in Dairying, Shinfield, Reading (Great Britain)

May 8th, 1964)

SUMMARY

I. The purpose of the investigation was to determine the extent to which the composition of the fatty acids in the a and /I positions of the glycerophosphatides and triglycerides in the livers of rabbits could be altered by the inclusion of different types of fat in the diet. 2. Five groups of rabbits were given diets cont&ing either 20% maize oil, 24% butter, 20% butterfat, ~0% hydrogenated coconut oil or 0.8% maize oil. After 30 weeks the animals were killed and the lipids were extracted from the livers. PooIed samples of pure phosphatidylcho~e, ph~p~tidylet~ol~~e and triglyceride were prepared from the liver lipids of each group by chromatography on columns of silicic acid. The positional distribution of the various fatty acids in the glycerophosphatides was determined after degradation with phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) and that in the triglycerides after partial hydrolysis with pancreatic iipase (glycerol ester hydrolase, EC 3.1.x.3). 3. In alI three glycerolipids there was a distinct tendency for stearic and palmitic acids to occur mainly in the a or a,a’ positions whereas the unsaturated acids together with my&tic acid occurred mainly in the @positions. In phosphatidylchohne, the proportion of saturated acids in the a position and the proportion of unsaturated acids in the @position were generally greater than in the corresponding positions in phosphatidylethanolamine. 4. The proportion of saturated acids in the &position of ~osphatidyl~o~e and the propo&on of unsaturated acids in the /? positions of both glycerophosphatides were not altered to any extent by dietary treatment. The proportions of saturated acids in the a positions of the phosphatidylethanolamine and ‘in the &a’ and’ /I positions of the triglyceride were affected considerably by differences in diet. 5. The results are discussed in terms of the.relationships that possibly exist between the metabolic pathways that lead to the biosynthesis of these three lipids in the liver.

Bioc&iim. Bio@hys. Acta, g8 (1965) 1~x50

138

J. II. MOORE, D. L. WILLIAMS

INTRODUCTION The work of KENNEDY l** has clearly shown that the major metabolic pathways that lead to the biosynthesis of the glycerophosphatides and triglycerides are closely related. For instance, evidence from experiments in vitro has suggested that in animal tissuesl~* phosphatidylcholine, phosphatidylethanolamine and triglyceride are derivved from a common pool of a,Bdiglyderide. If the glycerophosphatides and triglycerides are derived from a common a,/?diglyceride precursor, then some adequate explanation must be sought to account for the characteristic differences in fatty acid composition that have been reported for the various glycerophosphatides and triglyceridesisolated from any one tissue (e.g. rat livera) and the differences in fatty acid composition of the same glycerophosphatide isolated from different tissues (e.g. lecithin from beef liver4 and beef spinal cord*). The differences in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine could be due to differences in the affinities of phosphorylcholine-glyceride transferase (CDPcholine: ~,zdiglyceride cholinephosphotransferase, EC 2.7.8.2) and phosphoryl ethanolamineglyceride transferase (CDPethanolamine : I,a-diglymride ethanolaminephosphotransferase, EC 2.7.8.x) for different a,&diglycerides. Alternatively the two glycerophosphatides may not in fact be derived from the same a&diglyceride poola. It seemed possible that a knowledge of the extent to which the patterns of fatty acid distribution in the various lipids of a particular tissue could be altered by dietary means might contribute to the elucidation of this problem. Accordingly, a detailed examination was made of the distribution of the various fatty acids in the a and /? positions of the three main glycerolipids (i.e. phosphatidylcholine, phosphatidylethanolamine and triglyceride) in the livers of rabbits that had been given diets differing widely in fatty acid composition. The results of this investigation are now reported. EXPERIMENTAL Rabbits and diets The rabbits used in this study were thirty New Zealand White males. The animals were 6 months of age at the beginning, of the experiment and were divided into 5 groups of 6 each. The five experimental diets were composed as follows: to 80 parts of a semipurified, low-fat basal diet was added 20 parts of maize oil for Group I, 24 parts of whole butter for Group 2, 20 parts of butterfat for Group 3, 20 parts of hydrogenated coconut oil for Group 4 and I part of maize oil and 42 parts of wheat starch for Group 5. After the rabbits had been given the experimental diets ad l&turn for 30 weeks they were killed by a blow on the head and as rapidly as possible thereafter the liver was removed from each animal. The nutritional aspects of this experiment have been reported in detail elsewhere’. Extraction of lipids The lipids were extracted from the livers by the technique of FOLCH et at.‘. After washing with 0.88% (w/v) KC1 the lipid extracts were taken to dryness under reduced pressure at room temperature by means of a rotary film evaporator attached to a supply of nitrogen. The-lipids were rapidly taken up in chloroform and the resulting solutions were filtered. Sufficient methanol was then added so that the lipids Biochim

Biophys.

Ada.

98 (1965) 137-150

STUDIESON THE GLYCEROLIPIDS OF RABBITLIVER

I39

could be stored in chloroform-methanol (2 : I, v/v). The lipid solutions were stored in the dark at 5” until they were analysed. Isolation

of phosphatidyldwline,

@os#atidylethanolamine

and

triglycefide

Lipid samples from each liver were chromatographed on 5-g columns of silicic acid (IOOmesh; A.R., Mallinckrodt Chemical Works) by an adaptation of the methods of ~&RINETTI et al.Oand HANAHANet aLlo described by MOOREANDDORAN~~. Crude cephahn fractions were eluted from the columns with chloroform-methanol, 4: I (v/v) and crude lecithin fractions were eluted with chloroform-methanol, 3 : 2 (v/v). The crude phosphatides obtained from the individual livers were then pooled to give a bulked sample of cephalin and a bulked sample of lecithin from each of the 5 groups of rabbits. The pooled samples of crude phosphatides were then re-chromatographed on columns of silicic acid (HANAHANet al.‘“) until each sample gave rise to one spot only, corresponding either with phosphatidylethanolamine or with phosphatidylcholine, when examined by thin-layer chromatography. Thin-layer chromatoplates of Silica Gel G (E. Merck A.G., Darmstadt, Germany)-ammonium sulphate (go : IO, w/w) with a solvent system of chloroform-methanol-water (65 : 25 : 5, v/v) were used for the examination of the phosphatidylcholine eluates (MANGOLD’*).Pure lecithin, prepared from egg-yolk lipid’8 was used as a reference standard. Thin-layer chromatoplates of Silica Gel G with a solvent system of chloroform-methanol-14 N ammonia(65 : 25 : 4, v/v) were used for the examination of the phosphatidylethanolamine eluates (HORROCKS l*).Efficient separation of phosphatidylethanolamine from phosphatidylserine is obtained with the latter solvent system. Synthetic L-a-dimyristoyl phosphatidylethanolamine (a gift from Professor ERICHBAERof the University of Toronto) was used as a reference standard. To isolate the liver triglycerides, samples of lipid were chromatographed on columns of silicic acid by an adaptation of the technique of BARRONANDHANAHANI5described by MOOREANDDORAN16.The efficiencies of the separations were checked by chromatography on thin-layer plates of Silica Gel G with a solvent system of light petroleum (boiling range, 4o6o”)diethylether (go : IO, v/v). Enzymic degradation

of the li$ids Samples of the two glycerophosphatides were hydrolysed with the phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) of rattlesnake (Crotahs adamentms) venom. Hydrolysis was continued until examination of portions of the reaction mixtures by thin-layer chromatography indicated that the release of fatty acids from the ,d positions was complete. The free fatty acids were then separated from the lysophosphatides by chromatography on columns of silicic acid. Again, thinlayer chromatography was employed to check the efficiency of the separations. The techniques involved in the degradation of the phosphatides have been described in detail by MOOREand WILLIAMS’*. The technique of enzymic degradation of the triglycerides was similar to that described by COLEMAN 17. Hydrolysis was carried out with a partially purified lipase (glycerol ester hydrolase, EC 3.1.1.3) preparation (a gift from Dr. A. DAVISON,Unilever Research Laboratories, Shambrook) that had been obtained from pig pancreas. The triglyceride samples (approx. 50 mg) were emulsified with 8 ml of 1.2 M ammonium chloride-ammonia buffer (pH 8.5) and 0.1 ml of a 25% (w/v) bile salt solution in a semi-micro homogenizer (Measuring and Scientific Equipment Ltd. London). Biochim.

Biofihys.

Ada,

g8 (1965)

137-150

=4o

J. H. MOORE, D. L. WILLIAMS

The bile salt solution was prepared from partially purified sodium taurocholate. Sodium taurocholate (laboratory reagent grade) was found to contain glyceride impurities when examined by thin-layer chromatography”‘. These impurities were removed by extracting an aqueous solution of sodium taurocholate with hexane. The partially purified bile acid was then obtained by extracting the acidified aqueous solution with diethyl ether. The enzyme solution was obtained by dissolving IO mg of the lipase preparation in a mixture of z ml of 1.2M ammonium chloride-ammonia buffer (pH 8.5) and 2 ml of 22% (w/v) CaCl, * 6H,O. The enzyme solution (4 ml) was added to 8.1 ml of emulsified substrate and the reaction was carried out at 37” in a vessel fitted with glass and calomel electrodes. The reaction mixture was maintained at pH 8.5 by the gradual addition of 7 N ammonia from an “Agla” micrometer syringe (Burroughs Wellcome & Co., London). A fine stream of nitrogen was passed through the reaction mixture to ensure efficient mixing. When about 50% of the total fatty acids had been liberated, the reaction was stopped by the addition of an amount of 4 N HCl suf?icient to bring the solution to pH I. The lipids were then removed from the acidified aqueous solution by several extractions with diethylether. The reaction products (i.e. mainly free fatty acids and @-monoglycerides) were separated by thinlayer chromatography. Chromatoplates of Silica Gel G were used with a solvent system of light petroleum (boiling range, 4o-6o”)diethylether-formic acid (60: 40: I, v/v).After development, the chromatograms were sprayed with 0.2% (w/v) dichlorofluorescein in methanol-water (g5:5, v/v) and the positions of the lipids were determined by viewing the plates under ultraviolet light. The bands of Silica Gel G containing the monoglycerides were transferred from the plates to glass microcolumns (20 mm x6 mm) and the lipids were eluted with chloroform-methanol (I :4, v/v). In the various procedures involved in thin-layer chromatography the manipulation of the lipid samples was carried out under nitrogen. Fatty acid analysis The fatty acids of the various lipid samples were converted to the corresponding methylesters by the method of STOFFEL et al. lo. As a routine procedure, the methyl esters were chromatographed on columns of 10% (w/w) Apiezon L grease (obtained from Edwards High Vacuum Ltd., Crawley, Sussex) on 10o-120 mesh Celite (obtained from J. J’s., Ewe& Surrey). The columns, 122 cm in length and 4 mm in internal diameter; were operated at 200’ with a gas flow of 60 ml/min in an Argon chromatograph (W .G. Bye Ltd., Cambridge) fitted with a B-ionization detector. To determine the proportion of linolenic and linoleic acids and to assist in the identification of peaks, certain of the samples of methyl esters were also chromatographed on columns of !ro% (w/w) polyethyleneglycol adipate on xoo-120 mesh Celite at 185’ with a gas flow of 60 mk/min. Major peaks on the reco,rder chart were identified by comparison of their retention units with those of the methyl esters of pure fatty acids (obtained from Calbiochem. Inc., New York). The fatty acid composition of each sample was calculated from the recorder tracing by the method of CARROLL”. RESULTS

The fatty acid compositions of the various dietary fats are given in Table I (the shorthand designation of FARQUBAR et al.*% is used to denote the various fatty (Bjochim. Biopbys. Acta, 98 (1965) 13P-150

STUDIES

ON THE GLYCEROLIPIDS

141

OF RABBIT LIVER

acids). As discussed elsewhere’, the differences in composition of the whole butter and butterfat were probably due to the method used for the large-scale separation of the butterfat from the whole butter during the preparation of the diets. D,etailed results for linoleic and linolenic acids cannot be given for the liver lipids since only certain of the methyl ester samples were analysed on both the polar andnon-polar columns, However, it appeared that linolenic acid occurred only to the extent of about 0.9% of the total C,, polyunsaturated acids in the lipid samples obtained from the rabbits of Groups I and 5 and about 6% of the total C,, pol~nsat~ated acids in the lipid samples obtained from the rabbits of Groups 2,3 and 4. The compositions of the total fatty acids in the phosphatidylcholine, phosphatidylethanolamine and triglyceride obtained from the livers of the rabbits on the five dietary treatments are given in Table II. It would appear that the proportion of saturated fatty acids in the phosphatidylcholine remained relatively constant (i.e. between 46 and 48%) in spite of large differences in the nature of the dietary fatty acids. In contrast, the proportion of saturated acids in the phosphatidyleth~ola~e varied considerably (i.e. between 44% for Group 4 and 56% for Group I) with dietary treatment. Surprisingly, this latter variation was completely opposite to the variation that might have been expected.The small excess of unsaturated acids in TABLE THE

I

FATTY

The fatty

ACID

COMPOSITION

acid composition

OF THE

VARIOUS

is expressed

DIETARY

FATS

as molar percentage

of the total.

Acid 12 : 0 Maize oil Whole butter Butterfat Hydrogenated

0

coconut oil

x4

:0

0

I.2

10.2

3-s 41.1

20.5 22.9

r6:o 1r.g

34.6 35.6 x3.9

r6:1

r8:o

0.3

I.4

2.0

12.2

2.6 0.3

8.0 18.6

I8

: I

26.3 29.6 io.2

1.4

x8 : 2

r8 : 3

58.4 3.3 1.6 0.7

I.6 0.4 0.6 0

all of the samples of phosphatidylcholine points to the presence of a certain proportion of molecules with unsaturated acids in both the x and @ positions. The rather anomalous effect of diet on the fatty acids of phosphatidyle~~ol~e is emphasized further when it is realized that in the livers of the rabbits in Group I there must have been an excess of phosphatidylethanolamine molecules .tith saturated acids in both the tc and /l positions and that in the livers of the rabbits in Group 4 there must have been an excess of molecules with unsaturated acids in both the DLand fi positions. The effects of diet on the individual fatty acids of the glycerophosphatides are more evident when the fatty acids of the Q and @ positions are considered separately, but it is clear from Table II that one of the major effects of diet was on the proportions of oleic and linoleic acids. There were only small amounts of lauric acid in the liver phosphatides of all groups but the level of myristic acid, also a relatively small component, was clearly increased by the inclusion of hydrogenated coconut oil in the diet. These findings are in close agreement with the results of MULDER& al-s” who found that when coconut oil was included in the diet of rabbits for a period of 40 weeks, the concentration of my&tic acid in the erythrocyte phosphatides was increased only to a slight extent whereas the concentration of lauric acid remained unaltered. It should be noted that comparatively small amounts of’ arachidonic Biochim.

Biof~hys. Acta, 98 (rg6$

137-150

S

v

z @ c” w

_$ rg m

Q a

II

OF THE

22.5

-._

:o

:I

: I

:3 :4

18

20

Total unsatd.

20

18 : z + 18 : 3

17 :1

16

Total satd.

~~

18

O-5

19.7

51-3

3.1

0.8

42.8

1.0


20.5

15.3

30.6

1.3 0.3

56.1

35.0

0.7

18.8

0.3

0.4 0.9

PE

0.4

I.1

47.8

23.2

: 0 : 0

16 17

0.3

:o

0.2 1.1

PC

15

I4 : 0 12 0

Acid

ACIDS

75.1

0.7


53.2

3.0

1.7

IS.7

29.7

24.6

48.8

2.5 0.6

46.3

19.0

25-4 0.3

0.5

0.2 0.9

52.2

1.9

32.8 13.5 0.5

2.7 0.8

46.9

20.5

0.6

24.1

0.2

0.3 1.2

55.4

0.5


4x-9 8.9

4.1 <0.x

42.5

2.4


34.5

0.1 55 40.1

51.0

2.7

‘0”:6”

26.0

1.6 0.7

48.2

'7-9

0.5

27-5

0.3 1.4 0.6

49.3

1.1

0.2

12.2

32.0

3.2 0.6

49.8

18.4

0.5

28.7

0.5 I.4 0.3

PE

49.1

0.5

(0.1

36.7 8.0


3.9

48.8

5.9


37.0

5”:; (0.1

TG

PC

TG

PC

PE

(20 o/o buttwfaf diet)

Gvowp 3

Gro@ a [a4 o/Qbutfer diet)


1.1

24.2

2.6

20.8

(0.X

0.1 0.7 CO.1

XG

oil diet)

PHOSPWATIDYLETHANOLAMINE

of the total. PC, phosphatidylcholine;

IN THE PHOSPHATIDYLCHOLINE,

is expressed as molar percentage

FATTY

Group I (20 oh m&e

The fatty acid composition

THE COMPOSITIONS

TABLE AND

TRIGLYCERIDR

OBTAINED

52.5

3.4

2.0

30.2 13.8

2.8 0.3

46.4

20.0

21.9 0.1

3.2 0.5 0.7

55.3

2.7

29.9 16.6 0.9

4.5 0.7

43.6

19.9

G-5

~9.2

3-1 0.5 0.4

38.9

o.S

(0.1

28.0 6.6

(0.1

3.8

59.2

6.4

to.1

‘4.7 1.5


51.3

2.9

23.8 21.2 0.5

2.6 0.3

47.7

IS.7

30.1 0.4

0.9 0.2 0.4

50.5

2.4

15.9 I.7

26.1

3.6 0.8

49.1

21.8

23-7 0.9

0.3 2.0 0.4

PE

PC

TG

PC

PE

G--W 5 (low-fat diet)

11.7

50.3

o-7


30.5


7.4

47.8

4.7


39.3


0.4 3.4

TG

TG, triglyceride.

FROMRABBIT LIVER

Group 4# (20 0/0 h@rogenated coconzlt oiE diei)

PE, phosphatidylethanolamine;

STUDIES

ON THE GLYCEROLIPIDS

OF RABBIT LIVER

‘43

acid and only traces of docosahexaenoic acid were present in the liver phosphatides of the rabbit and that these amounts were not increased by the presence of high levels of linoleic acid in the diet. In all groups there appeared to be a number of characteristic differences between the fatty acids of the liver phosphatides and those of the liver triglycerides. For instance, the levels of stearic acid in both phosphatides were considerably higher than the corresponding levels in the triglycerides. The compositions of the fatty acids in the a positions of the phosphatidylcholine and phosphatidylethanolamine isolated from the rabbit livers are given in Table III. The proportion of saturated acids in the a position of phosphatidylcholine was not altered appreciably by dietary treatment although there were noticeable differences in the individual fatty acids. In the LXposition in three of the phosphatidylcholine samples (Groups 2, 3 and 5) the concentration of palmitic acid was markTABLE

III

TRB coIPos,TIoNs BTRANOLAMINE

OF

THE

OBTAINED

FATTY FROM

Compo&on of the fatty PE, phosphatidylethanolamine.

The

ACIDS RABBIT

IN

THE

acids is expressed

croup I (a0 o/o maize oil

Cf POSITIONS

OF

PHOSPAATIDYLCHOLINE

as molar percentage

PC

:0

PE

of the total.

(a~;~J!/obutterfat

(10 yO hydrogenated coconut oil die:)

(low-fa! diet)

PC

PC

PC

PC

3

GouP

PE

PE

0.3

0.2

0.2

I.1

0.2

0.7

0.7

0.4 I.4

15 :o

0.6

0. I

0.9

0.2

I.1

39.7

23.6

43.7

32.5

47.4

40.3

1.0

1.0

1.1

58.0

33.1

34.0

I.1

30. I

29.

83.0

83.2

79.6

68.7

81.5

70.8

I.4

0.4 0.2 II.2

I.1 0.6 12.8

I.2 0.9 22.9

I.2 0.6 10.1

1.1 0.7 20.9

6.1 0.4

4.2 0.3

4.5

5.6

5.7

3.6

0.8

5.2

0.5

0.6

0.5

16.4

16.3

19.8

30.7

18.1

28.9

18

I.0

: 0

Total satd.

16 17 18 18 20

:I :I :I :2 :4

:::

+

18

:3

Total unsatd.

0.4 0.7

0.3 2.2

0.6


39.7 0.9 I

croup 5

4

PE

0.3

16 : o 17 :o

PC, phosphatidylcholine;

GwJ

14 :o

12

PHOSPHATIDYL

croup a (24 % buuN.wdiet)

diet) Acid

AND

LIVER

0.4 2.3 0.3

39.2
23.4

37.2

0.2

0.6

0.9 0.7

0.3

I.2

31.8

54.2 0.9 27.1

3:::

79.5

59.2

84.0

74.7

I.9
3.6

2.4

1.8

0.7

0.3

0.8

22.9

9.5

19.1

0.7

12.7 0.1

2.7

2.5

0.5

0.6

19.8

40.0

15.4

24.8

I.0

greater than that of stearic acid but in the remaining two samples (Groups and 4) the concentrations of these two saturated acids were about the same. The level of myristic acid in the a position of the phosphatidylcholine obtained from the rabbits of Group 4 was not as high as might have been predicted from the results given in Table II but nevertheless was higher than the corresponding levels for the other 4 groups. It should also be noted that the oleicacid: linoleic acid ratio in the a position of phosphatidylcholine appeared to be influenced to a small extent by the level of linoleic acid in the diet. The proportion of saturated acids in the a position of phosphatidylethanolamine varied considerably with dietary treatment i.e. from 59% for Group 4 (20% hydrogenated coconut oil diet) to 83% for Group I (20% maize oil diet). This rather extraordinary effect of diet on the fatty acids in the a position of phosphatidylethanolamine was brought about in Group I mainly by an edly I

Biochim.

PE

Biophys.

Acta. 98 (1965)

137-150

34.6

J. I-I. MOORE, D. L. WILLIAMS

144

elevated level of stearic acid and a decreased level of oleic acid. At present no explanation may be offered to account for this seemingly anomalous finding but it should be noted that somewhat similar results were in fact obtained by RHODES~ in a study of the effect of adding cod-liver oil to the diet of the laying hen on the composition of the egg-yolk phosphatides. After the hens had been given the diet containing codliver oil for 19 days a 17% decrease in the degree of unsaturation was observed for the fatty acids in the a position of the phosphatidylethanolamine isolated from the egg-yolk lipid. No such decrease in the degree of unsaturation occurred in the fatty acids in the a position of the phosphatidylcholine obtained from the egg yolks. In the a position of phosphatidylethanolamine obtained from the rabbits of Group 4 it was equally surprising to find that the concentrations of linoleic and palmitoleic acids were more than twice the corresponding values obtained for the other groups. TABLE IV THECOMPOSITIONS RTHANOLAMINE

OF THE

OBTAINED

FATTY FROM

ACIDS

RABBIT

IN

THE

b

POSITIONS

OF PIiOSPHATIDYLCIiOLINE

AND

PHOSPHATIDYL-

LIVER

composition of the fatty acids is expressedas molar percentageof the total. PC, phosphatidylcholine; PE, phosphatidylethanolamine.

The

12

0.2

I4 :o I5 :o

1.1

16 17 18 IS 20 20

:I :I :I :2 :3 :4

+

18

:3

Total unsatd.

~~~$~

PC

PC

PC

PC

diet)

:o

Total satd.

(20 yO hydrogenated coconut oil diet)

diet) PC

16 : o 17 :o 18 : o

Grou$ 3 (20 “/5butterfat diet)

Grou+

(20 0/O maizeoil

Acid

(24 % butter

GYOU~I

PE 0.5 I.7 0.6

2

PE

PE

Group4

PE

diet) PE

0.2

0.4

0.2

0.6

0.8

0.7

0.2

1.1

1.7

I.4

2.2

4.2

3.9

0.9

2.9

0.2

0.6

0.9

0.6

0.2

0.5


0.1 6.8
14.1 0.5 12.0

7.1

0.3 15.8

7.6

X7.9

4.7

15.1


0.1 7.0


0.2 7.7

0.1 2.8

0.1

8.1

4.3

7.0

12.9

29.4

13.8

25.3

15.2

29.2

13.5

28.5

11.7

23.8

0.8 0.5 22.4 55.2

2.3 0.5

2.0 0.6

4.2 0.8

2.1 0.8

5.3 0.6

3.8 0.7

5.4 0.8

2.9

28.2 36.9

47.9 27.2

42.7 21.9

41.9 33.2

43.1 18.7

46.9 24. I

36.9 20.5

0.4 38. I 39.8

1.6 5.9


2.5 5.2

1.0 3.3

I.3 4.8

0.4 1.8

4.1 6.1

1.9 5.3

5.4

5-4 0.8 33.1 29.3 2.5 4.3

84.1 69.9

86.0

70.8

87.5

75.4

86.4

69.7

0.2

85.4 73.9

6.1


1.0

When the results for the a position of phosphatidylcholine are compared with those for the a position of phosphatidylethanolamine, it would seem that certain fatty acid patterns, characteristic of each phosphatide, are maintained in spite of marked differences in the type and amount of fat in the diet. For instance, in all groups the concentration of pahnitic acid in phosphatidylcholine was greater than in phosphatidylethanolamine, but the concentration of oleic acid in phosphatidylethanolamine was greater than that in phosphatidylcholine. With the exception of Group I, the proportion of unsaturated acids in the phosphatidylethanolamine was greater than that in the phosphatidylcholine. The compositions of the fatty acids in the/l positions of the two glycerophosphatides obtained from the rabbit livers are given in Table IV. The proportion of unsaturated acids in the b position of phosphatidylcholine was not altered to any great Biochim. Biophys. Acta, g8 (1965)

137-150

12.9

0.5

STUDIES ON THE GLYCEROLIPIDS OF RABBIT LIVER

I45

extent by dietary treatment and varied only between 84 and 88%. In Groups 2, 3, 4 and 5 the oleic acid : linoleic acid ratios in the fi positions of phosphatidylcholine were 1.8, 1.3, I.9 and x.0, respectively, but in Group I (20% maize oil diet) this ratio decreased too.4r. Again, these results are in good agreement with the observations of MULDER et aLss who noted that the replacement of coconut oil by maize oil in the diet of rabbits resulted in a marked increase in the concentration of linoleic acid and a corresponding decrease in the concentration of oleic acid in the /? position of phosphatidylcholine and phosphatidylethanolamine isolated from the erythrocytes. As with phosphatidylcholine, the proportion of unsaturated acids in the p position of phosphatidylethanolamine did not vary to any extent (i.e. from 70 to 75%) with dietary treatment. The effect of diet on the levels of my&tic, oleic and linoleic acids in the /? positions was very similar for both glycerophosphatides. Irrespective of dietary treatment, there were certain characteristic differences between the fatty acids in the #? position of phosphatidylcholine and those in the B position of phosphatidylethanolamine. For instance, the proportion of saturated acids in the @ position of phosphatidylethanolamine was always greater than in the f3 position of phosphatidylcholine. i

b

’ :1

Fig. I. Gas chromatographic analysis (Apiezon L columns) of the fatty acids of (a) “a,a’-distearoyl, /?-mono-olein” and (b) the monoglyceride obtained by partial hydrolysis of the “a,a’-distearoyl,/J-mono-olein” with pancreatic lipase.

Comparison of the results given in Table III with those given in Table IV shows that in both liver phosphatides the unsaturated acids occurred mainly, but not exclusively, in the j? positions, whereas the saturated acids, with one exception, occurred mainly, but, again not exclusively, in the a positions. Although myristic acid was only a relatively small component of the liver phosphatides in Group 4 it should be noted that this acid occurred to a greater extent in the /3 position than in the a position in both glycerophosphatides. A similar distribution of myristic acid between the a and /l positions of the phosphatidylcholine isolated from buttermilk has been reported by HAWKE**. The efficiency of the technique used for the determination of triglyceride strncture is illustrated by an experiment with synthetic “a,a’xlistearoyl /I-monoolein” (also a gift from Dr. A. DAVISON). The fatty acids of the synthetic triglyceride are shown in the chromatogram reproduced in Fii. ra (the percentage composition was: stearic acid, 67.7; oleic acid, 30.4; palmitic acid, 1.9). The fatty acids of the /?-monoglyceride isolated from the reaction mixture are shown in the chromatogram Biochim.

Biophys.

Ada,

98 (1965) 137-150

J. H. MOORE, D. L. WILLIAMS

146

reproducedin Fig. rb (the percentage composition was : stearic acid, 4.4; oleic acid, 8g.g; pahnitic acid, 5.8). The percentage composition of the fatty acids in the a,a’ positions, calculated by the method of COLEMAN’?was therefore: stearic acid, 99.3; oleic acid, 0.7; palmitic acid, 0.0. As might be expected, the diglyceride fraction isolated from the reaction mixture contained about 50% oleic acid and 50% stearic acid. In agreement with COLEMANAND FULTON*4 it was found that considerable amounts of free glycerol were produced during the reaction and that the free fatty acids liberated during hydrolysis contained some oleic acid. As discussed by COLEMANANDFULTON~” and DESNUELLEAND SAVARY2s these findings could probably be attributed to the isomerization of a proportion of the resulting a,/?-diglycerides to a,a’-diglycerides and the subsequent cleavage of the ester bonds in the a and a’ positions. The compositions of the fatty acids in the a,#’ positions (calculated by the method of COLEMAN~‘)and p positions of the triglycerides isolated from the livers of the rabbits given the various experimental diets are presented in Table V. In all TABLE V THEco~~os1~10~soF OBTAINED

FROM

The composition

TIMEFATTYACIDSINTHE

(20 oh maiaeoil

diet) a,a’

I2 :o

IN THE

@ POSITION 0F THE

TRIGLYCERIDES

of the fatty acids is expressed as molar percentage of the total. Group I

Acid

a,a’ POSITION AND

RABBITLIVER

0.3 1.4

Group

a,a’

a,a’

B

3

(20 o/obutterfat

diet) B

0.4 6.4

Gvoup4

Group5

a,a’

a,a’

(20 o/Ohydrogenated coconut oil diet)

6.4

B

2.7

(low-fat diet)

0.0

B

I.3

14 :o 16 : o 18 : o

0.I

0.1

0.1

26.2 2.6

10.9 2.7

42.2 3.1

19.5 0.7

46.0 6.8

19.1 4.1

12.3 42.8 6.1

19.5 24.2 7.2

0.0 48.4 5.7

21.1 2.9

Total satd.

29.1

15.3

50.3

27.1

58.3

30.0

61.6

53.6

54. I

35.4

I.4

3.2 “2::

6.0 53.8 10.5

2.8 30.4 6.2

6.3 t:::

4.1 26.3 5.6

3.4 3;‘:

6.5

, 0.6

7.0 25.5 II.2

8.3 40.6 12.8

70.9

39.7

67.9

36.5

44.2

44.2

62.8

16 18 18 20

0.0

B

Group 2 (24 yObutter diet)

6.4

:I 1.0 :I 23.3 : 2 + 18 : 3 45.6 :4 0.5

Total unsatd.

70.4

26.8 55.1

1.1

84.4

4.9

0.4 47.7

6.8

0.6

groups, the concentration

5.4

6.3

I.4

0.5

of myristic acid in the /I position was greater than in the a,,a’ positions. A similar distribution of myristic acid was noted for the a and /3 positions of both phosphatides obtained from the livers of the rabbits in Group 4. In sharp contrast, the concentration of palmitic acid in the /? position was much less than that in the a,a’ positions. The distribution of stearic acid between the a,a’ and p positions was similar to that of palmitic acid in Groups 2, 3 and 5 only. The concentrations of oleic and linoleic acids in the /? position were greater than the corresponding concentrations in the a& positions of the liver triglycerides in all groups. With the exception of Group 4, the distribution of palmitoleic acid followed that of oleic and linoleic acids. Although the concentrations of arachidonic acid in the triglyceride fractions were small, the results suggested that the distribution of this acid was also similar to that of oleic and linoleic acids. Unlike the variation in the composition of the fatty acids in the a position of phosphatidylethanolamine, the composition of the fatty acids in the a,a’ and /? Biochim. Biophys. Acta, 98 (1965) 137-150

10.1

1.1

STUDIESON TBB GLYCEROLIPIDS OF RABBITLIVER

=47

positions of the liver triglycerides varied in a manner that might have been predicted from the com~si~on

of the dietary fatty acids. In every. group, the proportion of

unsaturated acids in the &,a positions of the triglycerides was considerablyante than that in the a position of phosphatidylcholine. In four of the groups, a similar but less marked difference was observed when the fatty acid composition of the ~,a’ positions of the triglycerides was compared with that of the dl position of phosphatidylethanolamine. Although there were certain exceptions (8.g. in Group I), the proportion of -saturated acids in the t!Iposition of the liver triglycerides tended to be somewhat less than that in the ,&positions of the phosphatides. The Ievel of linoleic acid in the diet appeared to exert a pronounced effect on the oleic acid : linoleic acid ratio in both the a,u’ and p positions of the liver triglycerides. DiSCUSSION The fatty acid compositions of the pho~hatidylcho~e,

PhoSphatidyle~~~

lamine (usually containing Some phosphatidylserine) and triglyceride isolated from liver tissue have been reported for the rata**, ox*, and mouse lil, and the results are in many respects similar to those obtained by us for the corresponding lipids of rabbit liver. For instance, lauric and my&tic acids were found to account for only a small proportion of the fatty acids of the liver ph~pha~d~ of the rat a and mouse W. The high levels of stearic acid in the liver phosphatides and low levels in the liver triglycerides seemed to be a characteristic feature common to the rat, ox and rabbit. On the other hand, pahnitic acid was a major fatty acid component of the three liver lipids in all four species examined. However, the concentrations of arachidonic and docosahexaenoic acids in the liver phosphatides of the rabbit were strikingly less than the ~~es~n~g concen~tions reported for the phosphatides of rat%&, mouse@’ and ox* liver. Low levels of C,, and C,, polyunsaturated acids in the total phospholipid fraction of rabbit liver have also been reported by EVANS et al.“’ and S~RLL et aLaa. The distribution of the various fatty acids between the a,&’ and @ positions of the triglycerides isolated from rabbit liver is similar to that found by HANAHAN d al.m for the triglycerides of rat and beef liver and to that found for the adiposetissue triglycerides of the COW~~~~,sheepw**l, horse%*1, and man*l. However, this distribution of fatty acids is by no means characteristic of all triglycerides of animal origin for entirely different distributions have been reported for the adipose-tissue triglycerides of the pig*VW and for the triglycerides obtained from the milk of the cow 88‘ It would seem apposite now to examine whether the findings of the present investigation are consistent with the current view on the mechanism of the biosynthesis of phosphatidylcholiue, phosphatidylethanolamine and triglyceride. According to KENNEDY*, a,@%glyceride is a common precursor in the synthesis of all three of these lipids and it seems likely that the fatty acids in the @ position of the diglyceride precursor must be predominantly unsaturated whereas those in the cx position must be predominantly saturated. This a, p-diglyceride may arise from two main synthetic pathways. In the first, phosphatidic acid, formed by the reaction of a-glycerophosphate with fatty acyl CoAa8, is dephosphorylated by phosphatidate phosphatase (EC 3.1.3.4)*~. In the second, the diglyceride is formed by the reaction Biociim. Bio$hys. Act&, 98 (1965) 137-150

148

J. H. MOORE, D. L. WILLIAMS

of ,9-monoglyceride with fatty acyl CoA 8s. The /?-monoglyceride may be exogenous in origin since it appears likely that dietary fat, after partial hydrolysis in the gut,, is absorbed as a micellar dispersion of B-monoglyceride, free fatty acids and bile salts8*. It is conceivable that the second pathway played a considerable part in the synthesis of diglyceride in the rabbits given the high-fat diets (Groups I, 2, 3, and 4). For instance, in the rabbits of Group I (given the 20% maize oil diet) it it possible that appreciable amounts of exogenous @-monolinolein were absorbed. and, after being acylated in the tc position (probably in the mucosa of the small intestiness), were utilized for phosphatide and triglyceride synthesis in the liver. Such a pathway could well explain the high levels of linoleic acid in the /I position of the three liver lipids in Group I. On the other hand, it is unlikely that the high levels. of oleic acid observed in the /? positions of the liver lipids in Groups 2 and 3 (24% butter diet and 20% butterfat diet, respectively) could have been ultimately due to. the absorption of large amounts of exogenous @ mono-olein, for MCCARTHYet al.82, have shown that oleic acid is present mainly in the a,a’ position of milk triglycerides. In Group 4 (hydrogenated coconut oil diet) and Group 5 (low-fat diet), the high levels of oleic acid in the /3 positions of the liver lipids certainly could not have been due to the absorption of exogenous /Lmono-olein. Thus it would seem that the pathway in which a-glycerophosphate and phosphatidic acid are successive intermediates must have played an important role in the synthesis of the a$-diglyceride precursor in the rabbits of Groups 2,3,4 and 5. Although the recent work of LANDS AND HARTZ? should not be overlooked, the positional distribution of the saturated and unsaturated acids in the liver lipids could be accounted for if the enzyme system catalysing the acylation of the /l position of a glycerophosphate had a greater affinity for the unsaturated acids (and myristic acid) than for palmitic and stearic acids and conversely if the enzyme systems responsible for the acylation of the a’ position of a-glycerophosphate and the a position of B-monoglyceride have a greater affinity for palmitic and stearic acids than for the unsaturated acids (and myristic acid). It is unreasonable to expect that these enzyme systems are completely specific and the resulting, a,/?-diglycerides would consist of all four possible types of molecule although the a-saturated, &unsaturated type would be the predominant component. The characteristic differences in the fatty acid composition of the phosphatidylcholine and phosphatidylethanolarnine may then be explained by postulating certain differences in the specificities of the enzymes that catalyse the reactions between the a$-diglyceride and cytidine diphosphate choline and cytidine diphosphate ethanolamine. Irrespective of dietary treatment, about 80% of the fatty acids in the a position of phosphatidylcholine were saturated and about 86% of the fatty acids in the /l position were unsaturated (Tables III and IV). This suggests that the phosphorylcholine-glyceride transferase has a very strong affinity for those diglycerides with saturated acids (particularly palmitic acid) in the a position and unsaturated acids (particularly linoleic acid) in the @ position but only a very weak affinity for diglycerides possessing the other three types of configurations. In spite of variations in the type and level of dietary fat given to the rabbits, the percentage of unsaturated acids in the p position of phosphatidylethanolamine was consistently less than that in the /? position of phosphatidylcholine. In addition, levels as low as 60% were observed for the proportion of saturated acids in the a position of phosphatidylethanolamine. Thus, although the specificities of the two transferases appeared to be similar, phosphorylBiochim. Biofihys. Acta, 98 (1965) 137-150

STUDIES

ON

THE CLYCEROLIPIDS OF RABBIT LIVER

ethanolamine-glyceride

149

transferase would seem to possess a greater affinity for those

diglycerides with saturated fatty acids in the /? position or in both positions and unsaturated acids in the a position or in both positions than did phosphorylcholineglyceride transferase. The enzyme system involved in the acylation of the a position of the a,bdiglyceride to form txiglycexide would appear to tolerate an even wider variety of diglycerides. In experiments in vitro with the microsomal fraction of chicken liver, WEISS et aLa were able to show that a$-dilaurin and a$dioctanoin could act as “acceptors” in triglyceride synthesis but not in phosphatidylcholine synthesis. Although the existence of more than one pool of a$-diglyceride cannot be precluded, it appears that the fatty acid composition and structure of the glycerophosphatides and triglycerides in the liver of the rabbit could well be explained in terms of the biosynthetic pathways put forward by KENNEDY lvZ. However, it must be remembered that acertain proportion of phosphatidylcholine may have been synthesized by the methylation of phosphatidylethanolamine~ and also that a certain proportion of the phosphatidylethanolamine may have been synthesized by the decarboxylation of phosphatidylserine *. In addition, it is possible that the characteristic distribution of saturated and unsaturated acids between the a and B positions of the phosphatides may have arisen in part from a redistribution of the fatty acids after the nitrogenous bases had been incorporated into the molecules (LANDS and HARTZ’), ACKNOWLEDGEMENTS

The authors wish to acknowledge

the support and encouragement

given by

Dr. S. K. KON and to thank Miss J. CARRINCI and Miss A. BROADBENT for their skilled assistance. This study was supported by a grant from the Butter Information Council. REFERENCES I E. P. KENNEDY. Fednation PYOC., 16 (x957) 847. E. P. KENNEDY, Fadc*ation.Pvoc., 20 (1961) 934. 3 G. S. GETZ, W. BARnEY, F. STIRPE, B. M. NOTTON AND A. RENSRAW,

2

I 76. 4 G. M.

B&hem.

J., 80 (@I)

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J. HI. MOORE, D. L. WILLIAMS

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