/ . B.ochem. 85, 1453-1459 (1979)
Akiko OSANAI and Toshio SAKAGAMI Department of Biochemistry, Sapporo Medical College, Chuou-ku, Sapporo, Hokkaido 060 Received for publication, December 16, 1978
Detailed analyses of phospholipids were performed using male and female rabbit hearts (body weight, 2.5-3.0 kg). No difference between male and female was detected in the lipid-phosphorus content in 1 g of wet tissues or in the composition of the phospholipids. The amounts of the diacyl-, alkenyl-acyl-, and alkyl-acyl-components in cholinephosphoglyceride (PC) were 57%, 40%, and 3%, respectively. The results were the same in both male and female. The amount of the diacyl type of ethanolaminephospholipid (PE) was higher than that of the alkenyl-acyl type in the male (55 %, 43 %), but the contents of these two components were the same in the female rabbit (49%). The amounts of the alkyl-acyl types were about 3 % in both the male and female. The main difference between the male and female rabbit hearts was in the amount of Cis: i and Ci8:2 acids in PC, i.e., more C\&: i than Cis : 2 was detected in the male, whereas the reverse was the case in the female. The fatty aldehyde compositions of the alkenyl-acyl type PC were a little different from each other. Less C16:0 and more Ci8:0 and Cis: 1 aldehyde were observed in the male than in the female, while no difference between male and female was observed in the fatty acid and fatty aldehyde compositions of PE. The fatty acid compositions at the 1- and 2-positions of diacyl- and alkenyl-acyl type PC and PE were analyzed. The fatty acid composition of the diacyl type PC at the 2-position and that of the alkenylacyl type were similar except that more Cis: 1 and less C20:4 were found in the former in both the male and female rabbits. Different ratios of Cis.-1 to Cis: 2 between the male and female were also detected in these two fractions. The fatty acid composition of diacyl type PE at the 2-position and that of the alkenylacyl type were similar except that more C18:0 was observed in the former than in the latter, and small but significant amounts of C20:5 and C22:3-6 were clearly present in the latter, in contrast to the former, in both the male and female. As regards the fatty acid composition of the diacyl type compounds at the 1-position, no difference was observed in PC between the male and female, while more C16:0 and less Cis: 0 was found in PE in the male than in the female. Abbreviations: PC, cholinephosphoglyceride; PE, ethanolaminephospholipid. Vol. 85, No. 6, 1979
1453
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
Compositions of Diacyl-, Alkenyl-Acyl-, and Alkyl-AcylGlycerylphosphorylcholine and -Ethanolamine in Male and Female Rabbit Hearts
1454
A. OSANAI and T. SAKAGAMI
MATERIALS AND METHODS CR-1 diet was purchased from Japan Clea Co. Bee venom phospholipase A, [EC 3.1.1.4] and methylesters of fatty acids (Cu: o> Ci6: o» Cis: o. C20:0. and C22:0) were obtained from Sigma Co. Fatty aldehydes (C12:0, Cu • 0, and Cis: 0) were the gift of Dr. T. Akino, Department of Biochemistry, Sapporo Medical College. Extraction and Separation of Phospholipids— Four male and four female adult albino rabbits (2.5-3.0 kg) fed a CR-1 diet were decapitated under ether anesthesia. The hearts were excised, washed with cold physiological saline solution and trimmed to remove fat, then weighed and chopped into small pieces. All operations were carried out on ice at 4°C. Lipids were extracted with 20 volumes of chloroform-methanol (2:1, v/v) for 5 h at room temperature. The mixtures were shaken constantly during extraction. The extract was purified by the method of Folch et al. (13). Separation of phospholipids was performed by thin-layer chromatography as described previously (14). Phosphatidic acid and cardiolipin were separated by the procedure of Skipski et al. (15). The phosphatidylcholine and -ethanolamine fractions, obtained by thin-layer chromatography on neutral Kiesel gel G using chloroform-methanol-water (65 :25 : 4 , by volume), were subjected to further analyses. The plates were sprayed with 2',7'dichlorofluorescein-methanol solution, then left under a stream of nitrogen gas for 30 s to remove the developing solvent. Each phospholipid was
then rapidly extracted from the plates. Determination of the Compositions of Diacyl-, Alkenyl-Acyl-, and Alkyl-Acyl-Glycerylphosphorylcholine and -Ethanolamine—Less than 1.6 mg of each phospholipid was dissolved in 0.2 ml of 1 % HgCl t in glacial acetic acid and hydrolyzed at 37°C for 45 min. After separation of the lysophospholipids from the mild acid-stable phospholipids by thin-layer chromatography, mild alkaline hydrolysis was carried out by the method of Dawson (16). The lipid-phosphorus contents of the acid-labile, alkali-labile, and acid- and alkali-stable fractions were determined. Gas-Liquid Chromatography—The total lipid extract of the CR-1 diet and the purified phosphatidylcholine and -ethanolamine fractions of the rabbit hearts were treated with BF3-methanol (17), and the resulting methylesters of the fatty acids and dimethylacetals of the fatty aldehydes were separated by neutral Kiesel gel G thin-layer chromatography (benzene was used as a developing solvent). The methylesters were analyzed by the method of Metcalfe et al. (17) and the dimethylacetals by the method of Morrison and Smith (18). The peaks in the chromatograms were identified by comparison with the retention times of known standards, and the values for aldehydes were calculated by the method of Farguhar (19). The positional distribution of fatty acids in the acid-stable fraction was investigated with bee venom phospholipase A, by the method of Yamada et al. (20). Lipid-phosphorus was determined by the method of Bartlett (21): the method of Parker and Peterson (22) was also occasionally used. Because significant amounts of lysophospholipids were lost while washing the extract from the plates, the determination of lipid-phosphorus without this procedure was required in some cases. Two animals were killed simultaneously and the lipid extracts were kept in a freezer at —20°C during the analyses, which were accomplished within 3 weeks. No change could be found in the compositions under the conditions described above. RESULTS AND DISCUSSION The analyses were repeated four times with the same heart fractions and the means and standard /. Biochem,
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
Several studies on the phospholipid composition of heart muscles have been carried out on humans and some species of animals, including the rabbit (1, 2). Although the fatty acid compositions of the main phospholipids have been investigated in the cow (3-8), pig (4, 9), rat (10, 11), and human (12), there are few reports describing the positional distribution of fatty acids in the different components of the choline- and ethanolaminephosphoglycerides (5). In the present paper the positional distribution of fatty acids and the composition of the fatty aldehydes of the phosphatidylcholine and -ethanolamine components were determined in male and female rabbit hearts.
COMPOSITION OF PHOSPHOLIPIDS IN RABBIT HEARTS
TABLE I. Phospholipid compositions of male and female rabbit hearts. Values are expressed as percentages of total phospholipid-P (n=S). Phospholipids
Male
Lysophosphatidylcholine Sphingomyelin Phosphatidylcholine Phosphatidylinositol Phosphatidylserine Phosphatidylethanolamine Phosphatidylglycerol Phosphatidic acid Cardiolipin
Female
0.8 ±0.5* 0.5 ±0.2 4. 3±0.5 3.8±0.8 38.4±2. 1 42. 2±2. 2 3.1 ±0.5 3.2±0.9 3. 3±0.5 3.4±0.6 34.0±2. 1 32.2±1.5 1.1 ±0.4 1.2±0.3 0.4 ±0.3 0.9±0. 5 14. 5 ±0.5 12.7±1.4
Owens (1), who did not mention the sex of the animals used. The diacyl-, alkenyl-acyl-, and alkyl-acylglycerylphosphorylcholine and -ethanolamine compositions are shown in Table II. The percent distribution of these three types among cholinephosphoglyceride in the male is the same as in the female. However, in the ethanolaminephospholipid, the percentage of the diacyl component is higher than that of the alkenyl-acyl component in the male, but the values of these components are the same in the female. The percentages of alkenyl-acyl components among these phospholipids in the female rabbit heart are almost the same as those found by Owens (1), who did not describe the alkyl-acyl components of these phospholipids. The alkyl-acyl components accounted for only 2.2-2.7% of the phospholipids in both sexes. There are few reports concerning the content of the alkyl-acyl components but many reports concerning the plasmalogen content in hearts (23). The alkyl-acyl component accounted for 5.2% or less of the cholinephosphoglyceride in bovine hearts (7, 24-26) and human heart (27), and 2 % and 3.6% of the ethanolaminephospholipid in bovine heart (26) and human heart (27), respectively. Table III shows the fatty acid composition of the total lipid extract from the CR-1 diet. The predominant fatty acid is Ci8:2 (41.2%), followed by Ciero (17.7%), C i 8 : i (17.6%), and Cis:3 (17.1%), Although fatty acids more unsaturated than tri-unsaturated were hardly detectable, about 80% of the total fatty acids consisted of unsaturated acids. Animals were fed the CR-1 diet for about 6 months after weaning.
» Means ± SD.
TABLE II. Diacyl-, alkenyl-acyl-, and alkyl-acylglycerylphosphorylcholine (PQ and -ethanolamine (PE) in male and female rabbit hearts (%). The determination of lipid-phosphorus after mild acid hydrolysis was performed by the method of Paker and Peterson (22). Values in parentheses are percentages of total phospholipid-P (/i=8). Male
Types
Female
PC
PE
PC
PE
Diacyl
58.9±3. 2i(22.6)
55.1 ±3. 2 (18.7)
56.7±1.4 (23. 9)
48. 8±3.7 (15.7)
Alkenyl-acyl
38.9±3.3 (H.9)
42.7±3. 7 (14.5)
40.7±2.3 (17.2)
48. 5±3.5 (15.6)
AJkyl-acyl • Means ± SD. Vol. 85, No. 6, 1979
2.2±0.5
(0.8)
2.2±0. 9 (0.7)
2.6±0.9
(1. 1)
2. 7±0.5
(0.9)
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
deviations were calculated. The values obtained with the other three male rabbit hearts fell within the same range of values. However, one of the four female rabbit hearts studied showed some differences from the other three. Values are presented mostly as the means of the values from two separate determinations of each of four rabbit hearts. However, in some cases, where n=6, the last two hearts were analyzed only once each. The amounts of lipid-phosphorus in 1 g of wet tissue in the male and female hearts were 666±59.8/ig 0 = 4 ) and 656±64.9/ig (/i=6), respectively. The compositions of phospholipids in the male and female rabbit hearts are shown in Table I. The differences between the two sexes are not significant. The composition of the phospholipids in the female is similar to that reported by
1455
1456
A. OSANAI and T. SAKAGAMI
Chain length : number of double bonds
Percentage of total fatty acids
14 0 16 0 16 1 18 0 18 1 18 2 18 3 20 0 Saturated fatty acids Unsaturated fatty acids Saturated/unsaturated
0. 8±0.1* 17. 7 ±0.2 2. 8±0.1 1.2±0. 1 17.6±0. 2 41.2±0.4 17. 1 ±0.4 1.6±0.3 21.3 78.7 0.27
Means ± SD.
Table IV shows the compositions of fatty acids in the phosphatidylcholine and -ethanolamine fractions of the male and female hearts. In the phosphatidylcholine fraction, 44% of the fatty acid is saturated and the main fatty acid is Ci6:o (34%); Q 8 : i (26%), C«:2 (19%), and Can (9%) are the main unsaturated fatty acids in the male. In the female, similar results were obtained, except that the percentages of Cis:i (19%) and Ci8.-2 (22%) are reversed. The fatty acid composition of phosphatidylethanolamine in the male is the same as that in the female. The major saturated acid is Ci8:0 (3537%) and the major unsaturated acid is C M : * (34-36%). Compared with the phosphatidylcholine fraction, phosphatidylethanolamine contained more Cis: o» C » : i, and unknown acid and less Ci6: o, Ci6 : 1, Cis: i, and Cis: 2 acids. Because of the small amounts of the alkylacyl components of these phospholipids (less than 3%), we found difficulty in analyzing these materials. As the diacyl component accounts for more than 95% of the acid-stable phospholipids, the acid-stable fractions are considered to correspond to the diacyl components tentatively. The positional distribution of fatty acids in the acid-stable
TABLE IV. Fatty acid compositions of phosphatidylcholine (PQ and -ethanolamine (PE) fractions from male and female rabbit hearts (%). Experimental conditions were the same as in Table 111 («=6). Chain length : number of double bonds 14 : 0 16 :br 16 :0 16 : 1 17 :0 unknown 18 :0 18 : 1 18 :2 18 :3 20 :3 20 :4 20 :5 Saturated fatty acids Unsaturated fatty acids Saturated/unsaturated
Male
Female
PC
PE
PC
PE
0.5±n.2» 2. 5±0. 5 34.1±Z2 Z7±0.4 0.4±0. 1 0.4±0. 2 5.6±1.4 25.9±1.8 18.5±1.0 0.6±0.4 tr 8.8±1.6 tr 43.5 56.5 0.77
0.7 ±0.3 tr 7. 2±0. 9 0.9±0. 3 0.3±0. 1 3.7±0.9 35. 1±4. 5 6. 6±0. 6 9.5±1.7 0.6±0.3
1.0±0.3 1.4±0.2 35. 5±3.8 Z4±0.3 0.6±0.2 tr 5. 4 ±0.8 19.4±3.4 2Z2±0. 5 0.7 ±0.4
0.3 ±0.2 tr 5.9±1.0 0.4±0.3 0.3 ±0.1 Z0±0.8 37.4±5.8 7.3±1.7 8.9±Z2 0. 2±0.1
34.1±Z4 1.3±1.0 47.0 53.0 0.89
11.4±4.3 tr 43.9 56.1 0.78
36.2±4.3 1.1 ±0.7 45.9 54.1 0.85
« Means ± SD. J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
TABLE m . Fatty acid composition of total lipid in the diet. Methyl esters of fatty acids were separated by Kiesel Gel G thin-layer chromatography (developing solvent, benzene) after treating the total lipid extract of the diet with sodium hydroxide then boron fluoride-methanol solution. Gas-liquid chromatography was carried out with a Hitachi gas chromatograph, model K-53, using 200x0.3 cm stainless steel columns packed with diethylene glycol succinate 15% (Shimalite 201), 60-80 mesh, AW. Analysis were performed at 198°C with a nitrogen gas flow rate of 60 ml/min (/i=4).
1457
COMPOSITION OF PHOSPHOLIPIDS IN RABBIT HEARTS
female (Table V). On the other hand, no difference was observed between the sexes as regards the fatty aldehyde composition of the phosphatidalethanolamine (Table VI). Over 8 5 % of the aldehydes in both phosphatidalcholine and -ethanolamine was saturated; the main fatty aldehyde was Ci6:o (70-80%), in the former, and Cis:o (70%) in the latter. The fatty acid compositions of phosphatidalcholine in male and female rabbit hearts were similar, except that more Cis: I acid was found in the male than in the female. The content of Cis: 2 was the same in the male and female (Table V).
TABLE V. Compositions of fatty aldehydes and positional distribution of fatty acids of alkenyl-acyl- and diacylglycerylphosphorylcholine in male and female rabbit hearts (%). Gas-liquid chromatography of dimethyl acetals was carried out with a Hitachi gas chromatograph, model K-53 using 100x0.3 cm stainless steel columns packed with Apiezon M 25% (Shimalite 101), 60-80 mesh. Analyses were performed at 197°C with a nitrogen gas flow rate of 75 ml/min. Experimental conditions for methylesters of fatty acid were the same as in Table i n (n=6). Diacyl-glycerylphosphorylcholine
Alkenyl-acyl-glycerylphosphorylcholine Fatty aldehyde Male
Female
14 0 15 0
1.4±0. 8»
1.0±0. 6
16 br 80. 3 ±4. 5
Fatty acid
1-Position
2-Position
Male
Female
Male
Female
Male
Female
0.5 ±0.3
1.3 ±0.3
0. 2±0.1
0.4±0.2
0.4±0. 2
0.7±0.1
0.2±0.1
1.0±0.3
0.5 ±0.2
0.6±0.2
0.4±0. 1
0.4 ±0.2
0.3 ±0.1
0.2±0.1
0.1 ±0.1
l.l±0.6
0. 1±
0. 1±
13. 0±2. 8
19.2±4.8
71.2±0. 8
67.5±6.2
14. 5 ±4. 8
15.0±1.0
16 0
70. 0±3. 8
16 1
1.5±0.6
1.6±0. 4
2.2±1.0
5. O i l . 3
5.0±1.6
4.7±0.8
2.1 ±0.6
2. 5±0.4
17 0
1.3±0.4
2.1±0. 6
0.4 ±0.1
0.9±0.2
1.4±0.7
1. 2±0.5
0. 1±
0.4 ±0.2
(17 br) (6.6±0. 5)
(3.3±1. 2) 0.3 ±0.1
0.6±0. 2
tr
0.3 ±0.1
0. 1±
0.4 ±0.2
3.3±1.9
4.2±2.6
12.1±2.4
13.9±2.0
1.8±0.5
2. 1 ±0. 3
Unknown 18 0
6.6±1.6
3.4±1. 4
18 : 1
7.9±2.0
4.0±l. 7
29. 3±2. 1
21.4±2.5
7.9±1.4
8.6±1.1
39.9±4.4
30. 8 ±4.0
27.2±3.0
26.9 ±4.9
1.6±0.6
1.7±0. 8
27.3±1.1
33. 1±2.3
0.6±0.2
0.4 ±0.1
0.4±0. 1
0.7 ±0.2
0.2±0. 1
0. 2±0. 1
20 : 1
0.3 ±0.2
tr
18 : 2 18 : 3
4.7±1.0
4.3±1. 0
20 : 0 20: 2
2.5±1.9
1.1 ±0.6
2.1 ±0.5
0.6±0. 3
20: 3
1.1 ±0.5
1.9±0.4
1.8±0.2
0. 2±0. 1
20 : 4
17.6±2.4
15.6±2.2
8.8±1.8
1Z8±O. 3
20: 5
1.2±0.6
0.3 ±0.2
tr
tr
0.22
0.38
0.21
0.23
S/US"
6.09
9.10
Means ± SD. » Saturated fatty acid/unsaturated fatty acid. Vol. 85, No. 6, 1979
5.90
5.67
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
fractions was determined using bee venom phospholipase At, with a recovery of more than 98 % of the lipid-phosphorus as lysophospholipids after hydrolysis of both phospholipids. The compositions of the fatty aldehydes and positional distribution of the fatty acids of the alkenyl-acyl- (acid-labile) and diacyl- (acid stable) glycerylphosphorylcholine and -ethanolamine are shown in Tables V and VI, respectively. The fatty aldehyde compositions of the phosphatidalcholine in male and female rabbit heart are slightly different. In the male, less Ci6: o and more Cis: o and Q s : 1 were found than in the
1458
A. OSANAI and T. SAKAGAMI glycerylphosphorylcholine (70%) and Cis:o in diacyl-glycerylphosphorylethanolamine (66-73 %). In conclusion, differences between the sexes were detected in the fatty acid and fatty aldehyde compositions of cholinephosphoglyceride but little difference was detected in the case of ethanolaminephospholipid. Considering the results of other authors, the fatty acid compositions of the phospholipids of rabbit heart presented resemble those of the corresponding phospholipids in bovine (3, 4, 8), pig (•*, 9), and human (12) hearts. The results for the rat (10, 11) differ from those for other species. The fatty aldehyde compositions of phospholipids
TABLE VI. Compositions of fatty aldehydes and positional distribution of fatty acids of alkenyl-acyl- and diacylglycerylphosphorylethanolamine in male and female rabbit hearts (%). Conditions of gas-liquid chromatography were the same as in Table V (n=6). Alkenyl-acyl-glycerylphosphorylethanolamine Chain length: number of
Fatty aldehyde
Fatty acid
Diacyl-glycerylphosphorylethanolamine 1-Position
2-Position
Honhlc Vv^nHc
Male
Female
14 0 15 0
1.4±0. 7»
1.1 ±0. 7
16 br 16 0 16
17 0
br) Unk nown (17
18 0 18
40. 1±5.4 42. 6 ±3. 2
1
1
tr
1.8±0.6 4. 1±1. 4 (Z9±1.5) (1.5±0. 8) 33. 1 ± Z 4 35. 2±2. 3 15.9±0.4 12.4±1. 4
18 2 18
3
20
2
20
3
tr
tr
4.8±2.5
3. 1±1. 2
20 4 20
5
22
3
22 4 22
5
22
6
S/US"
3.83
Means ± SD.
b
5.45
Male 1.6±0. 5 0.5 ±0.2 0.3 ±0.1 9.7±3.4 Z6±1.0 0.5 ±0.2 0.4±0. 2 5.7±Z1 7.8±1.4
Female
Male
0.6±0.2 1.0±0.6 0.3 ±0.1 0. 2±0. 1 0.1± 0.6±0. 2 9.3±1.5 11.8±Z2 1.7±0.6 2. 7±0.8 0.4 ±0.2 0.7 ±0.4
Female
Male
Female
0.1
0.8 ±0.1
0.1
0.4±0. 1 0.2 ±0.1 8.6±Z4
0. 6±0. 2 0.3 ±0.1 0. 2±0. 1 8.2±1.8
Z0±0.4 0.8 ±0.1
1.6±0. 8 0.5 ±0.1
0.7 ±0.2 7.7±0. 3 1.2±0. 3 0.8 ±0.3
0.3 ±0.1 0.7 ±0.4 1.3±0.4 0.3 ±0.1 0. 2±0. 1 6.9±Z7 66.4±3. 5 73. 3±2. 3 20. 6 ±4. 4 14.0±5.9 6.1±1.7 13. 5±0.5 12. 3 ±0.7 7. 8±0.7 7. 1 ±0.4 1Z3±4. 1 Z 4 ± 1 . 3 2.5±1.0 14.0±2. 1 13.8±4.5 0.2±0. 1 0.1 0.3 ±0.1 1.2±0. 2 0.7±0. 2 0.7±0. 2 0.6±0. 2 0.9±0.4 1.7±0.5 41.5±Z0 48. 6±3.9 48.3±5.9 Z6±0.6 1.5±0.4 1.8±0. 3
12.5±Z6 0.3±0. 1 0.4±0. 2 1.1 ±0.6 48.4±3. 1 3.3±0.9 0.8±0.4 1.9±0.7 3.3±0.7 Z2±Z1 Z 9 ± 0 . 9 0.23
1.7±0.4 0.22
0. 6±0. 1
4.38
5.25
0.51
0. 8±0.3 0.32
Saturated fatty acid/unsaturated fatty acid. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
A reversed ratio of Cis •. 1 to Qs.- 2 in the male and female was also noted in diacyl-glycerylphosphorylcholine at the 2-position (Table V). No difference between the male and female was observed as regards the fatty acid composition of diacyl-glycerylphosphorylethanolamine at the 2-position, or in phosphatidalethanolamine. The fatty acid compositions of diacyl-glycerylphosphorylcholine at the 1-position were almost the same in the male and female, whereas that of diacyl-glycerylphosphorylethanolamine at the 1position consisted of more Ci6:o and less Ci8:o in the male than in the female. The predominant saturated acid at the 1-position is C16:0 in diacyl-
COMPOSITION OF PHOSPHOLIPIDS IN RABBIT HEARTS
It has also been found that changes in fatty acid composition occur at various stages in development and depend on the diet. The reason for the difference in the fatty acid Vol. 85, No. 6, 1979
composition of phosphatidylcholine between male and female is not known. REFERENCES 1. Owens, K. (1966) Biochem. J. 100, 313-361 2. Simon, G. (1970) Lipids 4, 607-614 3. Wheeldon, L.W., Schumert, Z., & Turner, D.A. (1965) /. LipidRes. 6, 481^89 4. Gray, G.M. (1960) Biochem. J. 77, 82-91 5. Schmid, H.H.O. & Takahashi, T. (1968) Biochim. Biophys. Ada 164, 141-147 6. Viswanathan, C.V., Phillips, F., & Lundberg, W.O. (1968) /. Chromatog. 35, 66-71 7. Viswanathan, C.V., Phillips, F., & Lundberg, W.O. (1968) J. Chromatog. 38, 267-273 8. Fleischer, S. & Rouser, G. (1965) / . Am. Oil Chem. Soc. 42, 588-607 9. Comte, J., Malsterrena, B., & Gautheron, D.C. (1976) Biochim. Biophys. Ada 419, 271-284 10. Dewailly, P., Sezille, P., Nouvelot, A., Fruchart, i.C, & Jaillard, J. (1977) Lipids 12, 301-306 11. Gloster, J. & Harris, P. (1970) Cardiovascular Res. 4, 1-5 12. Gloster, J. & Harris, P. (1969) Cardiovascular Res. 3, 45-51 13. Folch, J., Lees, M., & Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509 14. Osanai, A. & Sakagami, T. (1977) / . Biochem. 81, 1651-1659 15. Skipski, V.P., Barklay, M., Reichman, E.S., & Good, J.J. (1967) Biochim. Biophys. Ada 137, 80-89 16. Dawson, R.M.C. (1960) Biochem. J. 75, 45-53 17. Metcalfe, L.D., Schmitz, A.A., & Pelka, J.R. (1966) Anal. Chem. 38, 514-515 18. Morrison, W.R. & Smith, L.M. (1964) /. Lipid Res. 5,600-608 19. Farguhar, J.W. (1962) J. Lipid Res. 3, 21-30 20. Yamada, K., Imura, K., Taniguchi, M., & Sakagami, T. (1976) /. Biochem. 79, 809-817 21. Bartlett, G.R. (1959) /. Biol. Chem. 234, 466-468 22. Parker, F. & Peterson, N.F. (1965) / . Upid Res. 6, 455-460 23. Horrocks, L.A. (1972) in Ether Lipids (Snyder, F., ed.) pp. 177-272, Academic Press, New York 24. Pugh, E.L., Kates, M., & Hanahan, D.J. (1977) J. Lipid Res. 18, 710-716 25. Curstedt, T. (1977) Biochim. Biophys. Ada 489, 79-88 26. Pietruszko, R. & Gray, G.M. (1962) Biochim. Biophys. Ada 56, 232-239 27. Panganamale, R.V., Horrocks, L.A., Geer, J.C., & Cornwell, D.G. (1971) Chem. Phys. Lipids 6, 97-102 28. Spener, F. & Mangold, H.K. (1969) /. Lipid Res. 10, 609-613 29. Rao, P.V., Ramachandran, S., & Cornwell, D.G. (1967) J. Lipid Res. 8, 380-390 30. Warner, H.R. & Lands, W.E.M. (1963) /. Lipid Res. 4, 216-220
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
of rabbit heart are most similar to those of the pig (3, 4) and resemble those of the cow (4-7, 25, 29, 30) and human (27, 28), except that significant amounts of Cis: 3 aldehyde are present in both phospholipids in rabbit but not in bovine and human hearts. Identification of the Ci8:3 aldehyde has not yet been possible. Values of the ratio of the retention time of this aldehyde to that of the Cis: 0 aldehyde are 0.698-0.703. The positional distribution of fatty acids with diacyl components was investigated in bovine heart by Schmid and Takahashi (5), and the results are similar to those of the present study. The fatty acid composition at the 1-position has also been reported in the rat (10), but the predominant fatty acid in cholinephosphoglyceride was Cis: 0, the same as that in ethanolaminephospholipid. Although the fatty acid composition of phosphatidalcholine has also been reported for the cow (4, 5, 7, 25) and pig (4) and that of phosphatidalethanolamine in the cow (4-6), there is only one report (5) which compares the results for diacyl components at the 2-position and for the alkyl-acyl components. The difference between the fatty acid compositions of alkenyl-acyl- and diacyl-glycerylphosphorylcholine at the 2-position in rabbit heart is the same as that in the cow (5). The alkenyl-acyl components contain less Cis: 1 and more C20:4 than the diacyl components. The difference between the fatty acid compositions of these components at the 2-position of ethanolaminephospholipid in rabbit heart differs from that in bovine heart (5). In rabbit, the alkenylacyl component contains less Ci8:o than the diacyl component, whereas in the cow, the alkenylacyl component contains more Cis: 2 and less C M U than the diacyl component (5). The most distinctive result in the cow (5) is the high content of C20H (73%) in the diacyl component at the 2-position and the low content of this acid (27%) in the alkyl-acyl component. In rabbit heart, the content of C20: t in the diacyl component is very similar to that in the alkenyl-acyl component (4148%). The fatty acid composition of the alkylacyl component was not analyzed in the present study.
1459
Akiko OSANAI and Toshio SAKAGAMI Department of Biochemistry, Sapporo Medical College, Chuou-ku, Sapporo, Hokkaido 060 Received for publication, December 16, 1978
Detailed analyses of phospholipids were performed using male and female rabbit hearts (body weight, 2.5-3.0 kg). No difference between male and female was detected in the lipid-phosphorus content in 1 g of wet tissues or in the composition of the phospholipids. The amounts of the diacyl-, alkenyl-acyl-, and alkyl-acyl-components in cholinephosphoglyceride (PC) were 57%, 40%, and 3%, respectively. The results were the same in both male and female. The amount of the diacyl type of ethanolaminephospholipid (PE) was higher than that of the alkenyl-acyl type in the male (55 %, 43 %), but the contents of these two components were the same in the female rabbit (49%). The amounts of the alkyl-acyl types were about 3 % in both the male and female. The main difference between the male and female rabbit hearts was in the amount of Cis: i and Ci8:2 acids in PC, i.e., more C\&: i than Cis : 2 was detected in the male, whereas the reverse was the case in the female. The fatty aldehyde compositions of the alkenyl-acyl type PC were a little different from each other. Less C16:0 and more Ci8:0 and Cis: 1 aldehyde were observed in the male than in the female, while no difference between male and female was observed in the fatty acid and fatty aldehyde compositions of PE. The fatty acid compositions at the 1- and 2-positions of diacyl- and alkenyl-acyl type PC and PE were analyzed. The fatty acid composition of the diacyl type PC at the 2-position and that of the alkenylacyl type were similar except that more Cis: 1 and less C20:4 were found in the former in both the male and female rabbits. Different ratios of Cis.-1 to Cis: 2 between the male and female were also detected in these two fractions. The fatty acid composition of diacyl type PE at the 2-position and that of the alkenylacyl type were similar except that more C18:0 was observed in the former than in the latter, and small but significant amounts of C20:5 and C22:3-6 were clearly present in the latter, in contrast to the former, in both the male and female. As regards the fatty acid composition of the diacyl type compounds at the 1-position, no difference was observed in PC between the male and female, while more C16:0 and less Cis: 0 was found in PE in the male than in the female. Abbreviations: PC, cholinephosphoglyceride; PE, ethanolaminephospholipid. Vol. 85, No. 6, 1979
1453
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
Compositions of Diacyl-, Alkenyl-Acyl-, and Alkyl-AcylGlycerylphosphorylcholine and -Ethanolamine in Male and Female Rabbit Hearts
1454
A. OSANAI and T. SAKAGAMI
MATERIALS AND METHODS CR-1 diet was purchased from Japan Clea Co. Bee venom phospholipase A, [EC 3.1.1.4] and methylesters of fatty acids (Cu: o> Ci6: o» Cis: o. C20:0. and C22:0) were obtained from Sigma Co. Fatty aldehydes (C12:0, Cu • 0, and Cis: 0) were the gift of Dr. T. Akino, Department of Biochemistry, Sapporo Medical College. Extraction and Separation of Phospholipids— Four male and four female adult albino rabbits (2.5-3.0 kg) fed a CR-1 diet were decapitated under ether anesthesia. The hearts were excised, washed with cold physiological saline solution and trimmed to remove fat, then weighed and chopped into small pieces. All operations were carried out on ice at 4°C. Lipids were extracted with 20 volumes of chloroform-methanol (2:1, v/v) for 5 h at room temperature. The mixtures were shaken constantly during extraction. The extract was purified by the method of Folch et al. (13). Separation of phospholipids was performed by thin-layer chromatography as described previously (14). Phosphatidic acid and cardiolipin were separated by the procedure of Skipski et al. (15). The phosphatidylcholine and -ethanolamine fractions, obtained by thin-layer chromatography on neutral Kiesel gel G using chloroform-methanol-water (65 :25 : 4 , by volume), were subjected to further analyses. The plates were sprayed with 2',7'dichlorofluorescein-methanol solution, then left under a stream of nitrogen gas for 30 s to remove the developing solvent. Each phospholipid was
then rapidly extracted from the plates. Determination of the Compositions of Diacyl-, Alkenyl-Acyl-, and Alkyl-Acyl-Glycerylphosphorylcholine and -Ethanolamine—Less than 1.6 mg of each phospholipid was dissolved in 0.2 ml of 1 % HgCl t in glacial acetic acid and hydrolyzed at 37°C for 45 min. After separation of the lysophospholipids from the mild acid-stable phospholipids by thin-layer chromatography, mild alkaline hydrolysis was carried out by the method of Dawson (16). The lipid-phosphorus contents of the acid-labile, alkali-labile, and acid- and alkali-stable fractions were determined. Gas-Liquid Chromatography—The total lipid extract of the CR-1 diet and the purified phosphatidylcholine and -ethanolamine fractions of the rabbit hearts were treated with BF3-methanol (17), and the resulting methylesters of the fatty acids and dimethylacetals of the fatty aldehydes were separated by neutral Kiesel gel G thin-layer chromatography (benzene was used as a developing solvent). The methylesters were analyzed by the method of Metcalfe et al. (17) and the dimethylacetals by the method of Morrison and Smith (18). The peaks in the chromatograms were identified by comparison with the retention times of known standards, and the values for aldehydes were calculated by the method of Farguhar (19). The positional distribution of fatty acids in the acid-stable fraction was investigated with bee venom phospholipase A, by the method of Yamada et al. (20). Lipid-phosphorus was determined by the method of Bartlett (21): the method of Parker and Peterson (22) was also occasionally used. Because significant amounts of lysophospholipids were lost while washing the extract from the plates, the determination of lipid-phosphorus without this procedure was required in some cases. Two animals were killed simultaneously and the lipid extracts were kept in a freezer at —20°C during the analyses, which were accomplished within 3 weeks. No change could be found in the compositions under the conditions described above. RESULTS AND DISCUSSION The analyses were repeated four times with the same heart fractions and the means and standard /. Biochem,
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
Several studies on the phospholipid composition of heart muscles have been carried out on humans and some species of animals, including the rabbit (1, 2). Although the fatty acid compositions of the main phospholipids have been investigated in the cow (3-8), pig (4, 9), rat (10, 11), and human (12), there are few reports describing the positional distribution of fatty acids in the different components of the choline- and ethanolaminephosphoglycerides (5). In the present paper the positional distribution of fatty acids and the composition of the fatty aldehydes of the phosphatidylcholine and -ethanolamine components were determined in male and female rabbit hearts.
COMPOSITION OF PHOSPHOLIPIDS IN RABBIT HEARTS
TABLE I. Phospholipid compositions of male and female rabbit hearts. Values are expressed as percentages of total phospholipid-P (n=S). Phospholipids
Male
Lysophosphatidylcholine Sphingomyelin Phosphatidylcholine Phosphatidylinositol Phosphatidylserine Phosphatidylethanolamine Phosphatidylglycerol Phosphatidic acid Cardiolipin
Female
0.8 ±0.5* 0.5 ±0.2 4. 3±0.5 3.8±0.8 38.4±2. 1 42. 2±2. 2 3.1 ±0.5 3.2±0.9 3. 3±0.5 3.4±0.6 34.0±2. 1 32.2±1.5 1.1 ±0.4 1.2±0.3 0.4 ±0.3 0.9±0. 5 14. 5 ±0.5 12.7±1.4
Owens (1), who did not mention the sex of the animals used. The diacyl-, alkenyl-acyl-, and alkyl-acylglycerylphosphorylcholine and -ethanolamine compositions are shown in Table II. The percent distribution of these three types among cholinephosphoglyceride in the male is the same as in the female. However, in the ethanolaminephospholipid, the percentage of the diacyl component is higher than that of the alkenyl-acyl component in the male, but the values of these components are the same in the female. The percentages of alkenyl-acyl components among these phospholipids in the female rabbit heart are almost the same as those found by Owens (1), who did not describe the alkyl-acyl components of these phospholipids. The alkyl-acyl components accounted for only 2.2-2.7% of the phospholipids in both sexes. There are few reports concerning the content of the alkyl-acyl components but many reports concerning the plasmalogen content in hearts (23). The alkyl-acyl component accounted for 5.2% or less of the cholinephosphoglyceride in bovine hearts (7, 24-26) and human heart (27), and 2 % and 3.6% of the ethanolaminephospholipid in bovine heart (26) and human heart (27), respectively. Table III shows the fatty acid composition of the total lipid extract from the CR-1 diet. The predominant fatty acid is Ci8:2 (41.2%), followed by Ciero (17.7%), C i 8 : i (17.6%), and Cis:3 (17.1%), Although fatty acids more unsaturated than tri-unsaturated were hardly detectable, about 80% of the total fatty acids consisted of unsaturated acids. Animals were fed the CR-1 diet for about 6 months after weaning.
» Means ± SD.
TABLE II. Diacyl-, alkenyl-acyl-, and alkyl-acylglycerylphosphorylcholine (PQ and -ethanolamine (PE) in male and female rabbit hearts (%). The determination of lipid-phosphorus after mild acid hydrolysis was performed by the method of Paker and Peterson (22). Values in parentheses are percentages of total phospholipid-P (/i=8). Male
Types
Female
PC
PE
PC
PE
Diacyl
58.9±3. 2i(22.6)
55.1 ±3. 2 (18.7)
56.7±1.4 (23. 9)
48. 8±3.7 (15.7)
Alkenyl-acyl
38.9±3.3 (H.9)
42.7±3. 7 (14.5)
40.7±2.3 (17.2)
48. 5±3.5 (15.6)
AJkyl-acyl • Means ± SD. Vol. 85, No. 6, 1979
2.2±0.5
(0.8)
2.2±0. 9 (0.7)
2.6±0.9
(1. 1)
2. 7±0.5
(0.9)
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
deviations were calculated. The values obtained with the other three male rabbit hearts fell within the same range of values. However, one of the four female rabbit hearts studied showed some differences from the other three. Values are presented mostly as the means of the values from two separate determinations of each of four rabbit hearts. However, in some cases, where n=6, the last two hearts were analyzed only once each. The amounts of lipid-phosphorus in 1 g of wet tissue in the male and female hearts were 666±59.8/ig 0 = 4 ) and 656±64.9/ig (/i=6), respectively. The compositions of phospholipids in the male and female rabbit hearts are shown in Table I. The differences between the two sexes are not significant. The composition of the phospholipids in the female is similar to that reported by
1455
1456
A. OSANAI and T. SAKAGAMI
Chain length : number of double bonds
Percentage of total fatty acids
14 0 16 0 16 1 18 0 18 1 18 2 18 3 20 0 Saturated fatty acids Unsaturated fatty acids Saturated/unsaturated
0. 8±0.1* 17. 7 ±0.2 2. 8±0.1 1.2±0. 1 17.6±0. 2 41.2±0.4 17. 1 ±0.4 1.6±0.3 21.3 78.7 0.27
Means ± SD.
Table IV shows the compositions of fatty acids in the phosphatidylcholine and -ethanolamine fractions of the male and female hearts. In the phosphatidylcholine fraction, 44% of the fatty acid is saturated and the main fatty acid is Ci6:o (34%); Q 8 : i (26%), C«:2 (19%), and Can (9%) are the main unsaturated fatty acids in the male. In the female, similar results were obtained, except that the percentages of Cis:i (19%) and Ci8.-2 (22%) are reversed. The fatty acid composition of phosphatidylethanolamine in the male is the same as that in the female. The major saturated acid is Ci8:0 (3537%) and the major unsaturated acid is C M : * (34-36%). Compared with the phosphatidylcholine fraction, phosphatidylethanolamine contained more Cis: o» C » : i, and unknown acid and less Ci6: o, Ci6 : 1, Cis: i, and Cis: 2 acids. Because of the small amounts of the alkylacyl components of these phospholipids (less than 3%), we found difficulty in analyzing these materials. As the diacyl component accounts for more than 95% of the acid-stable phospholipids, the acid-stable fractions are considered to correspond to the diacyl components tentatively. The positional distribution of fatty acids in the acid-stable
TABLE IV. Fatty acid compositions of phosphatidylcholine (PQ and -ethanolamine (PE) fractions from male and female rabbit hearts (%). Experimental conditions were the same as in Table 111 («=6). Chain length : number of double bonds 14 : 0 16 :br 16 :0 16 : 1 17 :0 unknown 18 :0 18 : 1 18 :2 18 :3 20 :3 20 :4 20 :5 Saturated fatty acids Unsaturated fatty acids Saturated/unsaturated
Male
Female
PC
PE
PC
PE
0.5±n.2» 2. 5±0. 5 34.1±Z2 Z7±0.4 0.4±0. 1 0.4±0. 2 5.6±1.4 25.9±1.8 18.5±1.0 0.6±0.4 tr 8.8±1.6 tr 43.5 56.5 0.77
0.7 ±0.3 tr 7. 2±0. 9 0.9±0. 3 0.3±0. 1 3.7±0.9 35. 1±4. 5 6. 6±0. 6 9.5±1.7 0.6±0.3
1.0±0.3 1.4±0.2 35. 5±3.8 Z4±0.3 0.6±0.2 tr 5. 4 ±0.8 19.4±3.4 2Z2±0. 5 0.7 ±0.4
0.3 ±0.2 tr 5.9±1.0 0.4±0.3 0.3 ±0.1 Z0±0.8 37.4±5.8 7.3±1.7 8.9±Z2 0. 2±0.1
34.1±Z4 1.3±1.0 47.0 53.0 0.89
11.4±4.3 tr 43.9 56.1 0.78
36.2±4.3 1.1 ±0.7 45.9 54.1 0.85
« Means ± SD. J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
TABLE m . Fatty acid composition of total lipid in the diet. Methyl esters of fatty acids were separated by Kiesel Gel G thin-layer chromatography (developing solvent, benzene) after treating the total lipid extract of the diet with sodium hydroxide then boron fluoride-methanol solution. Gas-liquid chromatography was carried out with a Hitachi gas chromatograph, model K-53, using 200x0.3 cm stainless steel columns packed with diethylene glycol succinate 15% (Shimalite 201), 60-80 mesh, AW. Analysis were performed at 198°C with a nitrogen gas flow rate of 60 ml/min (/i=4).
1457
COMPOSITION OF PHOSPHOLIPIDS IN RABBIT HEARTS
female (Table V). On the other hand, no difference was observed between the sexes as regards the fatty aldehyde composition of the phosphatidalethanolamine (Table VI). Over 8 5 % of the aldehydes in both phosphatidalcholine and -ethanolamine was saturated; the main fatty aldehyde was Ci6:o (70-80%), in the former, and Cis:o (70%) in the latter. The fatty acid compositions of phosphatidalcholine in male and female rabbit hearts were similar, except that more Cis: I acid was found in the male than in the female. The content of Cis: 2 was the same in the male and female (Table V).
TABLE V. Compositions of fatty aldehydes and positional distribution of fatty acids of alkenyl-acyl- and diacylglycerylphosphorylcholine in male and female rabbit hearts (%). Gas-liquid chromatography of dimethyl acetals was carried out with a Hitachi gas chromatograph, model K-53 using 100x0.3 cm stainless steel columns packed with Apiezon M 25% (Shimalite 101), 60-80 mesh. Analyses were performed at 197°C with a nitrogen gas flow rate of 75 ml/min. Experimental conditions for methylesters of fatty acid were the same as in Table i n (n=6). Diacyl-glycerylphosphorylcholine
Alkenyl-acyl-glycerylphosphorylcholine Fatty aldehyde Male
Female
14 0 15 0
1.4±0. 8»
1.0±0. 6
16 br 80. 3 ±4. 5
Fatty acid
1-Position
2-Position
Male
Female
Male
Female
Male
Female
0.5 ±0.3
1.3 ±0.3
0. 2±0.1
0.4±0.2
0.4±0. 2
0.7±0.1
0.2±0.1
1.0±0.3
0.5 ±0.2
0.6±0.2
0.4±0. 1
0.4 ±0.2
0.3 ±0.1
0.2±0.1
0.1 ±0.1
l.l±0.6
0. 1±
0. 1±
13. 0±2. 8
19.2±4.8
71.2±0. 8
67.5±6.2
14. 5 ±4. 8
15.0±1.0
16 0
70. 0±3. 8
16 1
1.5±0.6
1.6±0. 4
2.2±1.0
5. O i l . 3
5.0±1.6
4.7±0.8
2.1 ±0.6
2. 5±0.4
17 0
1.3±0.4
2.1±0. 6
0.4 ±0.1
0.9±0.2
1.4±0.7
1. 2±0.5
0. 1±
0.4 ±0.2
(17 br) (6.6±0. 5)
(3.3±1. 2) 0.3 ±0.1
0.6±0. 2
tr
0.3 ±0.1
0. 1±
0.4 ±0.2
3.3±1.9
4.2±2.6
12.1±2.4
13.9±2.0
1.8±0.5
2. 1 ±0. 3
Unknown 18 0
6.6±1.6
3.4±1. 4
18 : 1
7.9±2.0
4.0±l. 7
29. 3±2. 1
21.4±2.5
7.9±1.4
8.6±1.1
39.9±4.4
30. 8 ±4.0
27.2±3.0
26.9 ±4.9
1.6±0.6
1.7±0. 8
27.3±1.1
33. 1±2.3
0.6±0.2
0.4 ±0.1
0.4±0. 1
0.7 ±0.2
0.2±0. 1
0. 2±0. 1
20 : 1
0.3 ±0.2
tr
18 : 2 18 : 3
4.7±1.0
4.3±1. 0
20 : 0 20: 2
2.5±1.9
1.1 ±0.6
2.1 ±0.5
0.6±0. 3
20: 3
1.1 ±0.5
1.9±0.4
1.8±0.2
0. 2±0. 1
20 : 4
17.6±2.4
15.6±2.2
8.8±1.8
1Z8±O. 3
20: 5
1.2±0.6
0.3 ±0.2
tr
tr
0.22
0.38
0.21
0.23
S/US"
6.09
9.10
Means ± SD. » Saturated fatty acid/unsaturated fatty acid. Vol. 85, No. 6, 1979
5.90
5.67
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
fractions was determined using bee venom phospholipase At, with a recovery of more than 98 % of the lipid-phosphorus as lysophospholipids after hydrolysis of both phospholipids. The compositions of the fatty aldehydes and positional distribution of the fatty acids of the alkenyl-acyl- (acid-labile) and diacyl- (acid stable) glycerylphosphorylcholine and -ethanolamine are shown in Tables V and VI, respectively. The fatty aldehyde compositions of the phosphatidalcholine in male and female rabbit heart are slightly different. In the male, less Ci6: o and more Cis: o and Q s : 1 were found than in the
1458
A. OSANAI and T. SAKAGAMI glycerylphosphorylcholine (70%) and Cis:o in diacyl-glycerylphosphorylethanolamine (66-73 %). In conclusion, differences between the sexes were detected in the fatty acid and fatty aldehyde compositions of cholinephosphoglyceride but little difference was detected in the case of ethanolaminephospholipid. Considering the results of other authors, the fatty acid compositions of the phospholipids of rabbit heart presented resemble those of the corresponding phospholipids in bovine (3, 4, 8), pig (•*, 9), and human (12) hearts. The results for the rat (10, 11) differ from those for other species. The fatty aldehyde compositions of phospholipids
TABLE VI. Compositions of fatty aldehydes and positional distribution of fatty acids of alkenyl-acyl- and diacylglycerylphosphorylethanolamine in male and female rabbit hearts (%). Conditions of gas-liquid chromatography were the same as in Table V (n=6). Alkenyl-acyl-glycerylphosphorylethanolamine Chain length: number of
Fatty aldehyde
Fatty acid
Diacyl-glycerylphosphorylethanolamine 1-Position
2-Position
Honhlc Vv^nHc
Male
Female
14 0 15 0
1.4±0. 7»
1.1 ±0. 7
16 br 16 0 16
17 0
br) Unk nown (17
18 0 18
40. 1±5.4 42. 6 ±3. 2
1
1
tr
1.8±0.6 4. 1±1. 4 (Z9±1.5) (1.5±0. 8) 33. 1 ± Z 4 35. 2±2. 3 15.9±0.4 12.4±1. 4
18 2 18
3
20
2
20
3
tr
tr
4.8±2.5
3. 1±1. 2
20 4 20
5
22
3
22 4 22
5
22
6
S/US"
3.83
Means ± SD.
b
5.45
Male 1.6±0. 5 0.5 ±0.2 0.3 ±0.1 9.7±3.4 Z6±1.0 0.5 ±0.2 0.4±0. 2 5.7±Z1 7.8±1.4
Female
Male
0.6±0.2 1.0±0.6 0.3 ±0.1 0. 2±0. 1 0.1± 0.6±0. 2 9.3±1.5 11.8±Z2 1.7±0.6 2. 7±0.8 0.4 ±0.2 0.7 ±0.4
Female
Male
Female
0.1
0.8 ±0.1
0.1
0.4±0. 1 0.2 ±0.1 8.6±Z4
0. 6±0. 2 0.3 ±0.1 0. 2±0. 1 8.2±1.8
Z0±0.4 0.8 ±0.1
1.6±0. 8 0.5 ±0.1
0.7 ±0.2 7.7±0. 3 1.2±0. 3 0.8 ±0.3
0.3 ±0.1 0.7 ±0.4 1.3±0.4 0.3 ±0.1 0. 2±0. 1 6.9±Z7 66.4±3. 5 73. 3±2. 3 20. 6 ±4. 4 14.0±5.9 6.1±1.7 13. 5±0.5 12. 3 ±0.7 7. 8±0.7 7. 1 ±0.4 1Z3±4. 1 Z 4 ± 1 . 3 2.5±1.0 14.0±2. 1 13.8±4.5 0.2±0. 1 0.1 0.3 ±0.1 1.2±0. 2 0.7±0. 2 0.7±0. 2 0.6±0. 2 0.9±0.4 1.7±0.5 41.5±Z0 48. 6±3.9 48.3±5.9 Z6±0.6 1.5±0.4 1.8±0. 3
12.5±Z6 0.3±0. 1 0.4±0. 2 1.1 ±0.6 48.4±3. 1 3.3±0.9 0.8±0.4 1.9±0.7 3.3±0.7 Z2±Z1 Z 9 ± 0 . 9 0.23
1.7±0.4 0.22
0. 6±0. 1
4.38
5.25
0.51
0. 8±0.3 0.32
Saturated fatty acid/unsaturated fatty acid. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
A reversed ratio of Cis •. 1 to Qs.- 2 in the male and female was also noted in diacyl-glycerylphosphorylcholine at the 2-position (Table V). No difference between the male and female was observed as regards the fatty acid composition of diacyl-glycerylphosphorylethanolamine at the 2-position, or in phosphatidalethanolamine. The fatty acid compositions of diacyl-glycerylphosphorylcholine at the 1-position were almost the same in the male and female, whereas that of diacyl-glycerylphosphorylethanolamine at the 1position consisted of more Ci6:o and less Ci8:o in the male than in the female. The predominant saturated acid at the 1-position is C16:0 in diacyl-
COMPOSITION OF PHOSPHOLIPIDS IN RABBIT HEARTS
It has also been found that changes in fatty acid composition occur at various stages in development and depend on the diet. The reason for the difference in the fatty acid Vol. 85, No. 6, 1979
composition of phosphatidylcholine between male and female is not known. REFERENCES 1. Owens, K. (1966) Biochem. J. 100, 313-361 2. Simon, G. (1970) Lipids 4, 607-614 3. Wheeldon, L.W., Schumert, Z., & Turner, D.A. (1965) /. LipidRes. 6, 481^89 4. Gray, G.M. (1960) Biochem. J. 77, 82-91 5. Schmid, H.H.O. & Takahashi, T. (1968) Biochim. Biophys. Ada 164, 141-147 6. Viswanathan, C.V., Phillips, F., & Lundberg, W.O. (1968) /. Chromatog. 35, 66-71 7. Viswanathan, C.V., Phillips, F., & Lundberg, W.O. (1968) J. Chromatog. 38, 267-273 8. Fleischer, S. & Rouser, G. (1965) / . Am. Oil Chem. Soc. 42, 588-607 9. Comte, J., Malsterrena, B., & Gautheron, D.C. (1976) Biochim. Biophys. Ada 419, 271-284 10. Dewailly, P., Sezille, P., Nouvelot, A., Fruchart, i.C, & Jaillard, J. (1977) Lipids 12, 301-306 11. Gloster, J. & Harris, P. (1970) Cardiovascular Res. 4, 1-5 12. Gloster, J. & Harris, P. (1969) Cardiovascular Res. 3, 45-51 13. Folch, J., Lees, M., & Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509 14. Osanai, A. & Sakagami, T. (1977) / . Biochem. 81, 1651-1659 15. Skipski, V.P., Barklay, M., Reichman, E.S., & Good, J.J. (1967) Biochim. Biophys. Ada 137, 80-89 16. Dawson, R.M.C. (1960) Biochem. J. 75, 45-53 17. Metcalfe, L.D., Schmitz, A.A., & Pelka, J.R. (1966) Anal. Chem. 38, 514-515 18. Morrison, W.R. & Smith, L.M. (1964) /. Lipid Res. 5,600-608 19. Farguhar, J.W. (1962) J. Lipid Res. 3, 21-30 20. Yamada, K., Imura, K., Taniguchi, M., & Sakagami, T. (1976) /. Biochem. 79, 809-817 21. Bartlett, G.R. (1959) /. Biol. Chem. 234, 466-468 22. Parker, F. & Peterson, N.F. (1965) / . Upid Res. 6, 455-460 23. Horrocks, L.A. (1972) in Ether Lipids (Snyder, F., ed.) pp. 177-272, Academic Press, New York 24. Pugh, E.L., Kates, M., & Hanahan, D.J. (1977) J. Lipid Res. 18, 710-716 25. Curstedt, T. (1977) Biochim. Biophys. Ada 489, 79-88 26. Pietruszko, R. & Gray, G.M. (1962) Biochim. Biophys. Ada 56, 232-239 27. Panganamale, R.V., Horrocks, L.A., Geer, J.C., & Cornwell, D.G. (1971) Chem. Phys. Lipids 6, 97-102 28. Spener, F. & Mangold, H.K. (1969) /. Lipid Res. 10, 609-613 29. Rao, P.V., Ramachandran, S., & Cornwell, D.G. (1967) J. Lipid Res. 8, 380-390 30. Warner, H.R. & Lands, W.E.M. (1963) /. Lipid Res. 4, 216-220
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1453/2185937 by University of West London user on 26 October 2018
of rabbit heart are most similar to those of the pig (3, 4) and resemble those of the cow (4-7, 25, 29, 30) and human (27, 28), except that significant amounts of Cis: 3 aldehyde are present in both phospholipids in rabbit but not in bovine and human hearts. Identification of the Ci8:3 aldehyde has not yet been possible. Values of the ratio of the retention time of this aldehyde to that of the Cis: 0 aldehyde are 0.698-0.703. The positional distribution of fatty acids with diacyl components was investigated in bovine heart by Schmid and Takahashi (5), and the results are similar to those of the present study. The fatty acid composition at the 1-position has also been reported in the rat (10), but the predominant fatty acid in cholinephosphoglyceride was Cis: 0, the same as that in ethanolaminephospholipid. Although the fatty acid composition of phosphatidalcholine has also been reported for the cow (4, 5, 7, 25) and pig (4) and that of phosphatidalethanolamine in the cow (4-6), there is only one report (5) which compares the results for diacyl components at the 2-position and for the alkyl-acyl components. The difference between the fatty acid compositions of alkenyl-acyl- and diacyl-glycerylphosphorylcholine at the 2-position in rabbit heart is the same as that in the cow (5). The alkenyl-acyl components contain less Cis: 1 and more C20:4 than the diacyl components. The difference between the fatty acid compositions of these components at the 2-position of ethanolaminephospholipid in rabbit heart differs from that in bovine heart (5). In rabbit, the alkenylacyl component contains less Ci8:o than the diacyl component, whereas in the cow, the alkenylacyl component contains more Cis: 2 and less C M U than the diacyl component (5). The most distinctive result in the cow (5) is the high content of C20H (73%) in the diacyl component at the 2-position and the low content of this acid (27%) in the alkyl-acyl component. In rabbit heart, the content of C20: t in the diacyl component is very similar to that in the alkenyl-acyl component (4148%). The fatty acid composition of the alkylacyl component was not analyzed in the present study.
1459
