Comp. Biochem. Physiol.. [,ol 62B. pp. 185 to 193 ~ PCr~lamtm Prexs Lid 1979 Pri~lted in Great Britain
0305-0491 79 0215-0185502./)0 0
SERUM AND LIVER LIPID COMPOSITION AND LECITHIN: CHOLESTEROL ACYLTRANSFERASE IN HORSES, EQUUS CABALLUS MUNEHIKO YAMAMOTO, YUKIO TANAKA a n d MICHIHIRO SUGANO l
Department of Chemistry, Kurume University School of Medicine, Mii-machi, Kurume 830 and 1Laboratory of Nutrition Chemistry, Department of Food Science and Technology, Kyushu University School of Agriculture, Ha.kozaki, Higashi-ku, Fukuoka 812, Japan
(Received 28 April 1978) Abstract--1. The lipid composition of serum and liver and some properties of serum lecithin:cholesterol acyltransferase of the horse were investigated. 2. Phospholipids and cholesterol were the major components of serum lipids and the concentration of triglyceride was considerably low. The concentration of liver lipids was comparable with that of other mammals. 3. Fatty acid composition of serum cholesterol ester resembled that of the 2-position of lecithin, except palmitic acid. 4. The activity of serum cholesterol esterifying enzyme was found to be 0.03-0.09/~mol/hr per ml. There was an equimolar decrease in free cholesterol and lecithin during incubation, and changes in unsaturated fatty acids in these two components were in good agreement. 5. Cholesterol esterification was reversibly inhibited by 5,5'-dithiobis-(2-nitrobenzoic acid). The acyltransferase had a specificity for linoleic acid.
INTRODUCTION
MATERIALS AND METHODS
There are numbers of reports concerning the lecithin:cholesterol acyltransferase (LCAT, E.C. 2.3.1.43) in plasma of human (Glomset, 1962; Glomset & Wright, 1964; Stokke & Norum, 1971; Marcel & Vezina, 1973; Wallentin & Vikrot, 1975; Wallentin, 1977) as well as of experimental animals commonly used as an animal model for atherosclerosis, monkeys (Sugano & Portman, 1964; Stokke, 1974; Mickel et al., 1975), dogs (Stokke, 1964; Mickel et al., 1975; Lacko et al., 1974), rabbits (Stefanovich, 1968; Wells & Rongone, 1969; Rose, 1972; Day & Proudlock, 1974), rats (Glomset, 1962; Sugano & Portman, 1965; Lacko etal., 1974) and chickens (Sugano etal., 1965, 1967; Sugano & Wada, 1967; Chinen, 1970a, b). Data are also available for calves (Noble etal., 1972), sheep (Noble et al., 1975) and some marine animals (Puppione, 1969). However, to date no reports have been published on the horse. Weik & Altmann (1971) described the fatty acid composition of serum lipids in gelding ponies. Morris e t a l . (1972) demonstrated that in the pony plasma more than 95% of phospholipids and more than 65% of cholesterol occurred in the lipoprotein fraction of d > 1.063, As far as we know, no additional information is available as to the lipid compositions of the horse serum and liver, except for the presence in depot fats of linolenic acid in large proportion (Brockerhoff etal., 1966). In this report we investigated some properties of L C A T in horse serum. The lipid compositions of serum and liver were simultaneously analyzed in detail. 185
Experimental animals Blood was obtained from the jugular vein of horses,
Equus cabullus, at the slaughterhouse in Kurume, Fukuoka. After sacrifice the liver was quickly excised, a portion of it was rinsed with isotonic saline and used for lipid analysis. Body weight, age and sex are listed in Table 1. The exact composition of the diet was not clear, but they had been fed the diets containing rice white bran, wheat bran, barley, defatted soy bean and corn in appropriate proportions. Male Wistar rats, Rattus norvegicus albus, fed a commercial pellet ration (Japan Clea, Tokyo, Rat Chow, CE-2) and weighing 300-500g served as blood donors. Fasting human blood was obtained from a healthy male adult (22 years old).
Lipid analyses Serum and liver lipids were extracted with chloroformmethanol (2:1, v/v) and purified (Folch et al., 1957). Cholesterol, lipid phosphorus, triglyceride and free fatty acids
Table 1. Body weight, age and sex of horse Horse No.
Body wt (kg)
Age (yr)
Sex
Class
1 2 3 4 5 6 7
400 500 540 500 400 450 650
6 3 3 3 2 7 6
Female Female Male Male Female Male Male
Light breed Half bred Half bred Light breed Light breed Light breed Light breed
186
MUNEHIKO YAMAMOTO,YUKIO TANAKA and MICHIHIRO SUGANO
were determined by the methods of Sperry & Webb (1950), Gomori (1942), Fletcher (1968) and Noma et al. (19731. respectively. Individual phospholipids were determined by the procedure of Rouser et al. (1966). Lecithin was hydrolyzed with snake venom phospholipase A2 (Crotalus adamanteus, Sigma Chemical Co.. St Louis) according to the method of Long & Penny t'1957/. Gas liquid chromatoqraphy (GLC) Fractionation of lipids was performed by TLC. Phospholipids were separated with chloroform methanol water (65:25:4, v/v) as a developing solvent and eluted with chloroform methanol-acetic acid water (50:39:1:10, v/v) (Arvidson, 1965). Cholesterol ester and triglyceride were chromatographed with petroleum ether-diethyl ether acetic acid (82:18:1, v/v) and eluted with chloroform methanol (2: 1. v/v) (lmaizumi et al., 1972). Free fatty acids obtained after saponification of each lipid with ethanolic potassium hydroxide were methylated by nitrosomethylurea (ICN Pharmaceuticals, Inc., Plainview) (Eneroth & Sj6vall, 1969). GLC was carried out using a gas chromatograph (Shimadzu, GC-6) with a hydrogen ionization detector. The column was 2.5 m x 3 mm (i.d.) packed with 1071, diethyleneglyco[ succinate polyester on chromosorb W, 8(~100mesh (Johns-Manville Sales Corp.. Denverl. The column and detector temperatures were 195 and 220C, respectively [lmaizumi et al.. 1972i. Measurement o1' serum LCA T actirit v (a) Tween 20 method. Serum (0.3 ml) was incubated with 0.03 ml of 0.5'!,, Tween 20 in a saline solution containing 0.061tCi 4-1*C-cholesterol (The Radiochemical Centre, Amersham, 53.7mCi/mmoll at 3 7 C (Sugano, 19711. The reaction was stopped by the addition of chloroform methanol (2:1, v:v). Cholesterol esters were separated into subclasses by TLC (Sugano & Portman, 1965). Each fraction was scraped into scintillation vials. The radioactivity was assayed by the liquid scintillation system (Beckman. LS-233) in toluene scintillator [0.2",, 2,5-diphenyloxazole and 0.005°~i 1,4-bis-(5-phenyloxazol-2-yl)-benzene] (Sigma Chemical Co., St Louis) and corrected for quenching by an external standard. (b) Common substra~e method, Sera from horse, human and rat were heated at 60'C for 45 min. The inactivated serum was incubated with acid washed Celite 545 coated with 2 llCi 4-~*C-cholesterol at 37 C for 5 hr (Portman & Sugano, 1964). After centrifugation, the supernatant was used as a common substrate. Substrate (0.3 ml) was incubated with enzyme serum (0.1 ml for horse and 0.05ml for human and rat) at 37 C. The radioactivity was determined as described above. RESUI,Ts
Serum lipid composition The concentration of serum lipids is shown in Table 2. Phospholipid was the most p r e d o m i n a n t lipid in horse serum, in which lecithin accounted for 73-83°,~ of total, while the concentration of cephalin and lysolecithin was extremely low. Cholesterol was the second p r e d o m i n a n t lipid, in which approx 80°~,, being the esterified form. The concentration of triglyceride and free fatty acids was also relatively low. As a result, cholesterol ester and lecithin constituted approx 65~o of total serum lipids. There were no apparent differences in the concentration and composition due to differences in sexes and ages. Fatty acid compositions o f serum lipids The fatty acid compositions of serum lipids are shown in Table 3. In cholesterol ester, percentage of
Table 2. Concentration of serum lipids Lipids Total cholesterol Free cholesterol Triglyceride Free fatty acids Total phospholipids Phosphatidylcholine Phosphatidylethanolamine Lysophosphatidylcholine Sphingomyelin Other phospholipids Total
Concentration 84.4 21.3 37.1 4.4 134 106 3.6 5.4 16.1 2.5 225
(76.1 96.0t 118.0 25.01 i26.1 45.0) (3.7 4.7) 1120 143i [91.7 115) (2.1 4.21 13.8 8.4~ t12.8 21.7) (0.8 3.7~ (238 2861
Values are the mean of five horses (range observed) and are expressed as mg/dl serum. Horse Nos 1 5 were used. linoleic acid was extraordinarily high. Palmitic acid, the second p r e d o m i n a n t fatty acid in this fraction, was measured by only 10--120o. In lecithin, linoleic acid was again the most predominant acid. The ratio of the saturated to the unsaturated acids was nearly equal. In the 1-position of lecithin, stearic acid was the most a b u n d a n t and the next. palmitic acid. the sum of the saturated acids being more than 900,,. Almost all of fatty acids in the 2-position of lecithin consisted of the unsaturated acids, especially linoleic acid. Palmitic acid was found only 3 4°,,. The fatty acid composition of lecithin resembled that of cholesterol ester, except for the difference in percentage of palmitic acid. Palmitic and stearic acids were the maior components of lysolecithin. This fraction was characterized by relatively high percentage of arachidonic acid. Although cephalin contained arachidonic acid at a high proportion, percentage of linoleic acid was considerably lower, and that of palmitic acid was higher than that of lecithin. In triglyceride, palmitic acid was thc largest c o m p o n e n t followed by oleic and linoleic acids in a decreasing order. Changes in lipid composition during incubation Changes in the concentration of major serum lipids during incubation at 3 7 C are given in Fig. 1. The concentration of free cholesterol decreased as time elapsed, the decrease for 2 4 h r being 49 64°,, (mean 58",, five horses). The rate of decrease remained linear for the first 2 hr incubation. The concentration of total lipid phosphorus, cephalin and sphingomyelin did not change, whereas the decrease in lecithin was at the equimolar rate to that of free cholesterol. The increase in lysolecitbin was equimolar to the decrease in lecithin during short time incubation, but became lower t h a n the decrease in lecithin after prolonged incubation. The concentration of free fatty acids increased progressively as the reaction proceeded and this increase was, in the molar ratio, about 3 times the decrease in triglyceride. Changes in the J~tty acid composition ~[ lysolecithin during incubation The fatty acid composition of serum lipids other t h a n lysolecithin remained unchanged during incubation for up to 24 hr. The changes in major fatty acids
O.9 (0.6-1.2)
1.5 (1.0-1.9)
0.5 (0.3-0.9)
15:0
31.8 (28.7-34.5)
30.5 (28.5-32.5)
3.4 (2.7-3.9)
2.3 (1.2 3.5)
2.2 (1.7 2.9)
1.7 (1.3--2.9)
2.O (1.3 2.9)
2.2 (1.4-3.1)
0.5 (0.4- 0.7) 1.3 (0.9 1.8)
0.6 (0.5-0.8) 0.7 (0.6-0.7) 0.5 (0.4-0.7)
13.6 (12.1-14.5) 23.8 (20.0-25.7) 3.1 (2.6-3.6)
23.1 (21.0-26.4)
0.5 (0.3 0.6)
17:0
1.8 (1.~2.1)
16:1
10.8 (10.0-11.7)
16:0
8.9 (7.7-10.0)
36.9 (35.8-38.2)
18.3 (15.4-20.6)
32.1 (30.7-33.6) 61.0 (58.3 65.3t 1.1 (0.4-1.8)
1.6 (1.4-1.9)
18:0
25.3 (22.6-27.8)
7.9 (7.1 9.4)
10.5 (8.3 12.4)
8.4 (7.4-8.9) 6.5 (5.6-6.9) 10.4 ~8.6-I 2.0)
7.8 (6.8 8.8)
Fatty acids 18:1
20.0 (15.1-22.5)
8.6 (7.0-9.8)
32.0 (27.8 35.6)
41.5 (40.9-42.5) 3.2 (2.5-3.8) 81.0 (79.0-82.5)
75.1 (74.0-76.7)
18:2
3.6 (1.5 5.4)
1.6 (1.2-2.4)
1.6 (1.3-2.3)
0.9 (0.6-1.3) 1.4 (1.2-2.0) 0.5 (0.2 0.7)
0.4 (0.2-0.8)
20:0
0.7 (0.5-0.8)
0.5 (0.3 O.6)
1.2 (0.9 1.6)
0.5 (0.3-0.6) 0.8 (0.6-1.0) 0.3 (0.2-0.4)
20:1
1.1 (O.8 1 . 3 )
0.8 (0.7 1.2)
0.4 (0.3-0.5) 0.5 (0.3-0.8) 0.6 (0.3-0.7)
20:2
1.1 (0.8-1.4)
0.6 (0.4-0.7)
0.3 (0.2 0.4)
20:3
1.7 (1.2-2.3)
6.5 (5.0-7.8)
5.6 (4.5 6.7)
1.0 (0.8 1.3) 0.9 (0.6-1.1) 2.0 (1.5-2.5)
0.9 (0.6-1.4)
20:4
Values are the mean of five horses (range observedt and are expressed as per cent of total fatty acids. Horse Nos 1 5 were used. Abbreviations: CE, cholesterol ester: PC, phosphatidylcholine: PE, phosphatidylethanolamine: LPC, lysophosphatidylcholine and TG, triglyceride.
1.0 (0.8-1.4) TG 2.0 (1.7-2.4)
1.4 (0.8-1.8) LPC
PE
2-position
l-position
0.7 (0.4-0.8) PC Total
CE
14:0
Table 3. Fatty acid composition of serum lipids
O
3
8
e"
,--i
188
MUNEH1KO YAMAMOTO, YUKIO TANAKA and MICHIHIRO SU(;ANO o 3L
~..,~ ~
CE LPC
6c
--~ o E
02 m 4c ,,= ol
/
FFA
::L .
~
0
x TC and TPL
3C
~' TG E
7o E ~
/
~ 5c N.
-- 80
,(3
-01
e -
-o3
o
/
t",,
I
0
1 2
I 4
--~ 2C o
/
/
/
/
/-
6o~
/,"
so gS
/
o+-
- 3 o g_~
Y
/
.¢.7. o
Z
_ 20 J3 '~_
~___.
P I~D- I ~ , , o m ii
J I 6 i2 Incubation time,
2
PC I FC 24 hr
Fig. 1. Time course of changes in the concentration of serum lipids during incubation, Values are the mean of five horses (Nos 1-5). Abbreviations: CE, cholesterol ester: FC, free cholesterol: TC, total cholesterol: FFA, free fatty acids: TG, triglyceride: TPL, total phospholipids: PC, phosphatidylcholine and LPC, lysophosphatidylcholine.
4
6
,: 12 Incubation time,
to c~ .E
24 hr
Fig. 3. Effect of incubation on the incorporation of 4-~4C cholesterol in Tween 20 into cholesterol esters and percentage distribution of radioactivity among individual esters. Values are the mean of three horses (Nos. 3 5). © O. total incorporation: • -O, saturated esters: • •. monoenoic: [] C], dienoic and U II, tetraenoic.
Stoichiometry of cholesterol ester!tication of lysolecithin during incubation are shown in Fig. 2. The proportion of stearic acid increased, while that of palmitic acid appeared to decrease slightly. Oleic, linoleic and arachidonic acids decreased to a lesser extent. Consequently, the fatty acid composition of lysolecithin after long time incubation resembled that of the l-position of lecithin (Table 3). Ester!fication qf 4J4C-cholesterol The results are given in Fig. 3. The rate of esterification of 14C-cholesterol in Tween 20 proceeded
linearly at least for 2 hr, in agreement with that demonstrated by chemical analysis (Fig. l). The distribution of radioactivity among individual cholesterol esters was constant irrespective of incubation periods and was similar to the fatty acid composition of that fraction (Table 3).
As shown in Fig. 4, the increase in m o n o u n s a t u r ated (mainly oleic acid) and diunsaturated (mainly linoleic acid) cholesterol esters during incubation was quantitatively compensated by the decrease in the corresponding acids of the 2-position of lecithin. In contrast, the increase in saturated fatty acids (mainly palmitic acid) in cholesterol ester was more t h a n twice the decrease in lecithin.
Rerersible inhibition qf the L C A T reaction by a SH blocking reagent The esterification was completely inhibited by an addition of 10 m M 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (Tokyo Chemical Industry Co.. Tokyo). The
C E - di ene.
02-zL
60--
*~ 50 ~
18:0
f :E_-a0 c
"o --
~
C E - said.
"5
~
C E - mono.
o
'
o
o
-~'
~
16:0
i§_0.,,
h
•
~
• P C - satd. • P C - mono.
--
\,
20--
._~ ~-02 '
2
4
'
6 12 Incubation time,
.
18:1
e
18:2
'~ 2 0 : 4 24 hr
Fig. 2. Time course of changes in the fatty acid composition of lysophosphatidylcholine. Values are the mean of five horses (Nos 1-5). Carbon chain length:numbers of double bonds.
o
•
I 2
L 4
I L 6 12 Incubation time,
PC-diene.
i 24 hr
Fig. 4. Quantitative analyses of fatty acids in cholesterol ester and phosphatidylcholine. Values are the mean of five horses (Nos 1 5). Satd, saturated fatty acids: Mona, monounsaturated and Diene, diunsaturated.
Serum and liver lipid composition in horses
189
higher t h a n that demonstrated in h u m a n and rat by the above authors.
40
Fatty acid specificity of cholesterol esterification ;7-
2o
0
I
2 3 4 Incubation
5 6 time,
9 hr
Fig. 5. Effect of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) on the LCAT activity and the reactivation by 2-mercaptoethanol. Serum (0.3ml) was incubated with 0.03 ml of a Tween 20 solution containing 0.06/~Ci 4-~4C cholesterol and 0.03 ml of DTNB in 0.1 M potassium phosphate buffer (pH 7.1) at 37°C. After incubation for 3 hr, 0.3 ml of mercaptoethanol was then added at the concentrations shown below. O-----
0305-0491 79 0215-0185502./)0 0
SERUM AND LIVER LIPID COMPOSITION AND LECITHIN: CHOLESTEROL ACYLTRANSFERASE IN HORSES, EQUUS CABALLUS MUNEHIKO YAMAMOTO, YUKIO TANAKA a n d MICHIHIRO SUGANO l
Department of Chemistry, Kurume University School of Medicine, Mii-machi, Kurume 830 and 1Laboratory of Nutrition Chemistry, Department of Food Science and Technology, Kyushu University School of Agriculture, Ha.kozaki, Higashi-ku, Fukuoka 812, Japan
(Received 28 April 1978) Abstract--1. The lipid composition of serum and liver and some properties of serum lecithin:cholesterol acyltransferase of the horse were investigated. 2. Phospholipids and cholesterol were the major components of serum lipids and the concentration of triglyceride was considerably low. The concentration of liver lipids was comparable with that of other mammals. 3. Fatty acid composition of serum cholesterol ester resembled that of the 2-position of lecithin, except palmitic acid. 4. The activity of serum cholesterol esterifying enzyme was found to be 0.03-0.09/~mol/hr per ml. There was an equimolar decrease in free cholesterol and lecithin during incubation, and changes in unsaturated fatty acids in these two components were in good agreement. 5. Cholesterol esterification was reversibly inhibited by 5,5'-dithiobis-(2-nitrobenzoic acid). The acyltransferase had a specificity for linoleic acid.
INTRODUCTION
MATERIALS AND METHODS
There are numbers of reports concerning the lecithin:cholesterol acyltransferase (LCAT, E.C. 2.3.1.43) in plasma of human (Glomset, 1962; Glomset & Wright, 1964; Stokke & Norum, 1971; Marcel & Vezina, 1973; Wallentin & Vikrot, 1975; Wallentin, 1977) as well as of experimental animals commonly used as an animal model for atherosclerosis, monkeys (Sugano & Portman, 1964; Stokke, 1974; Mickel et al., 1975), dogs (Stokke, 1964; Mickel et al., 1975; Lacko et al., 1974), rabbits (Stefanovich, 1968; Wells & Rongone, 1969; Rose, 1972; Day & Proudlock, 1974), rats (Glomset, 1962; Sugano & Portman, 1965; Lacko etal., 1974) and chickens (Sugano etal., 1965, 1967; Sugano & Wada, 1967; Chinen, 1970a, b). Data are also available for calves (Noble etal., 1972), sheep (Noble et al., 1975) and some marine animals (Puppione, 1969). However, to date no reports have been published on the horse. Weik & Altmann (1971) described the fatty acid composition of serum lipids in gelding ponies. Morris e t a l . (1972) demonstrated that in the pony plasma more than 95% of phospholipids and more than 65% of cholesterol occurred in the lipoprotein fraction of d > 1.063, As far as we know, no additional information is available as to the lipid compositions of the horse serum and liver, except for the presence in depot fats of linolenic acid in large proportion (Brockerhoff etal., 1966). In this report we investigated some properties of L C A T in horse serum. The lipid compositions of serum and liver were simultaneously analyzed in detail. 185
Experimental animals Blood was obtained from the jugular vein of horses,
Equus cabullus, at the slaughterhouse in Kurume, Fukuoka. After sacrifice the liver was quickly excised, a portion of it was rinsed with isotonic saline and used for lipid analysis. Body weight, age and sex are listed in Table 1. The exact composition of the diet was not clear, but they had been fed the diets containing rice white bran, wheat bran, barley, defatted soy bean and corn in appropriate proportions. Male Wistar rats, Rattus norvegicus albus, fed a commercial pellet ration (Japan Clea, Tokyo, Rat Chow, CE-2) and weighing 300-500g served as blood donors. Fasting human blood was obtained from a healthy male adult (22 years old).
Lipid analyses Serum and liver lipids were extracted with chloroformmethanol (2:1, v/v) and purified (Folch et al., 1957). Cholesterol, lipid phosphorus, triglyceride and free fatty acids
Table 1. Body weight, age and sex of horse Horse No.
Body wt (kg)
Age (yr)
Sex
Class
1 2 3 4 5 6 7
400 500 540 500 400 450 650
6 3 3 3 2 7 6
Female Female Male Male Female Male Male
Light breed Half bred Half bred Light breed Light breed Light breed Light breed
186
MUNEHIKO YAMAMOTO,YUKIO TANAKA and MICHIHIRO SUGANO
were determined by the methods of Sperry & Webb (1950), Gomori (1942), Fletcher (1968) and Noma et al. (19731. respectively. Individual phospholipids were determined by the procedure of Rouser et al. (1966). Lecithin was hydrolyzed with snake venom phospholipase A2 (Crotalus adamanteus, Sigma Chemical Co.. St Louis) according to the method of Long & Penny t'1957/. Gas liquid chromatoqraphy (GLC) Fractionation of lipids was performed by TLC. Phospholipids were separated with chloroform methanol water (65:25:4, v/v) as a developing solvent and eluted with chloroform methanol-acetic acid water (50:39:1:10, v/v) (Arvidson, 1965). Cholesterol ester and triglyceride were chromatographed with petroleum ether-diethyl ether acetic acid (82:18:1, v/v) and eluted with chloroform methanol (2: 1. v/v) (lmaizumi et al., 1972). Free fatty acids obtained after saponification of each lipid with ethanolic potassium hydroxide were methylated by nitrosomethylurea (ICN Pharmaceuticals, Inc., Plainview) (Eneroth & Sj6vall, 1969). GLC was carried out using a gas chromatograph (Shimadzu, GC-6) with a hydrogen ionization detector. The column was 2.5 m x 3 mm (i.d.) packed with 1071, diethyleneglyco[ succinate polyester on chromosorb W, 8(~100mesh (Johns-Manville Sales Corp.. Denverl. The column and detector temperatures were 195 and 220C, respectively [lmaizumi et al.. 1972i. Measurement o1' serum LCA T actirit v (a) Tween 20 method. Serum (0.3 ml) was incubated with 0.03 ml of 0.5'!,, Tween 20 in a saline solution containing 0.061tCi 4-1*C-cholesterol (The Radiochemical Centre, Amersham, 53.7mCi/mmoll at 3 7 C (Sugano, 19711. The reaction was stopped by the addition of chloroform methanol (2:1, v:v). Cholesterol esters were separated into subclasses by TLC (Sugano & Portman, 1965). Each fraction was scraped into scintillation vials. The radioactivity was assayed by the liquid scintillation system (Beckman. LS-233) in toluene scintillator [0.2",, 2,5-diphenyloxazole and 0.005°~i 1,4-bis-(5-phenyloxazol-2-yl)-benzene] (Sigma Chemical Co., St Louis) and corrected for quenching by an external standard. (b) Common substra~e method, Sera from horse, human and rat were heated at 60'C for 45 min. The inactivated serum was incubated with acid washed Celite 545 coated with 2 llCi 4-~*C-cholesterol at 37 C for 5 hr (Portman & Sugano, 1964). After centrifugation, the supernatant was used as a common substrate. Substrate (0.3 ml) was incubated with enzyme serum (0.1 ml for horse and 0.05ml for human and rat) at 37 C. The radioactivity was determined as described above. RESUI,Ts
Serum lipid composition The concentration of serum lipids is shown in Table 2. Phospholipid was the most p r e d o m i n a n t lipid in horse serum, in which lecithin accounted for 73-83°,~ of total, while the concentration of cephalin and lysolecithin was extremely low. Cholesterol was the second p r e d o m i n a n t lipid, in which approx 80°~,, being the esterified form. The concentration of triglyceride and free fatty acids was also relatively low. As a result, cholesterol ester and lecithin constituted approx 65~o of total serum lipids. There were no apparent differences in the concentration and composition due to differences in sexes and ages. Fatty acid compositions o f serum lipids The fatty acid compositions of serum lipids are shown in Table 3. In cholesterol ester, percentage of
Table 2. Concentration of serum lipids Lipids Total cholesterol Free cholesterol Triglyceride Free fatty acids Total phospholipids Phosphatidylcholine Phosphatidylethanolamine Lysophosphatidylcholine Sphingomyelin Other phospholipids Total
Concentration 84.4 21.3 37.1 4.4 134 106 3.6 5.4 16.1 2.5 225
(76.1 96.0t 118.0 25.01 i26.1 45.0) (3.7 4.7) 1120 143i [91.7 115) (2.1 4.21 13.8 8.4~ t12.8 21.7) (0.8 3.7~ (238 2861
Values are the mean of five horses (range observed) and are expressed as mg/dl serum. Horse Nos 1 5 were used. linoleic acid was extraordinarily high. Palmitic acid, the second p r e d o m i n a n t fatty acid in this fraction, was measured by only 10--120o. In lecithin, linoleic acid was again the most predominant acid. The ratio of the saturated to the unsaturated acids was nearly equal. In the 1-position of lecithin, stearic acid was the most a b u n d a n t and the next. palmitic acid. the sum of the saturated acids being more than 900,,. Almost all of fatty acids in the 2-position of lecithin consisted of the unsaturated acids, especially linoleic acid. Palmitic acid was found only 3 4°,,. The fatty acid composition of lecithin resembled that of cholesterol ester, except for the difference in percentage of palmitic acid. Palmitic and stearic acids were the maior components of lysolecithin. This fraction was characterized by relatively high percentage of arachidonic acid. Although cephalin contained arachidonic acid at a high proportion, percentage of linoleic acid was considerably lower, and that of palmitic acid was higher than that of lecithin. In triglyceride, palmitic acid was thc largest c o m p o n e n t followed by oleic and linoleic acids in a decreasing order. Changes in lipid composition during incubation Changes in the concentration of major serum lipids during incubation at 3 7 C are given in Fig. 1. The concentration of free cholesterol decreased as time elapsed, the decrease for 2 4 h r being 49 64°,, (mean 58",, five horses). The rate of decrease remained linear for the first 2 hr incubation. The concentration of total lipid phosphorus, cephalin and sphingomyelin did not change, whereas the decrease in lecithin was at the equimolar rate to that of free cholesterol. The increase in lysolecitbin was equimolar to the decrease in lecithin during short time incubation, but became lower t h a n the decrease in lecithin after prolonged incubation. The concentration of free fatty acids increased progressively as the reaction proceeded and this increase was, in the molar ratio, about 3 times the decrease in triglyceride. Changes in the J~tty acid composition ~[ lysolecithin during incubation The fatty acid composition of serum lipids other t h a n lysolecithin remained unchanged during incubation for up to 24 hr. The changes in major fatty acids
O.9 (0.6-1.2)
1.5 (1.0-1.9)
0.5 (0.3-0.9)
15:0
31.8 (28.7-34.5)
30.5 (28.5-32.5)
3.4 (2.7-3.9)
2.3 (1.2 3.5)
2.2 (1.7 2.9)
1.7 (1.3--2.9)
2.O (1.3 2.9)
2.2 (1.4-3.1)
0.5 (0.4- 0.7) 1.3 (0.9 1.8)
0.6 (0.5-0.8) 0.7 (0.6-0.7) 0.5 (0.4-0.7)
13.6 (12.1-14.5) 23.8 (20.0-25.7) 3.1 (2.6-3.6)
23.1 (21.0-26.4)
0.5 (0.3 0.6)
17:0
1.8 (1.~2.1)
16:1
10.8 (10.0-11.7)
16:0
8.9 (7.7-10.0)
36.9 (35.8-38.2)
18.3 (15.4-20.6)
32.1 (30.7-33.6) 61.0 (58.3 65.3t 1.1 (0.4-1.8)
1.6 (1.4-1.9)
18:0
25.3 (22.6-27.8)
7.9 (7.1 9.4)
10.5 (8.3 12.4)
8.4 (7.4-8.9) 6.5 (5.6-6.9) 10.4 ~8.6-I 2.0)
7.8 (6.8 8.8)
Fatty acids 18:1
20.0 (15.1-22.5)
8.6 (7.0-9.8)
32.0 (27.8 35.6)
41.5 (40.9-42.5) 3.2 (2.5-3.8) 81.0 (79.0-82.5)
75.1 (74.0-76.7)
18:2
3.6 (1.5 5.4)
1.6 (1.2-2.4)
1.6 (1.3-2.3)
0.9 (0.6-1.3) 1.4 (1.2-2.0) 0.5 (0.2 0.7)
0.4 (0.2-0.8)
20:0
0.7 (0.5-0.8)
0.5 (0.3 O.6)
1.2 (0.9 1.6)
0.5 (0.3-0.6) 0.8 (0.6-1.0) 0.3 (0.2-0.4)
20:1
1.1 (O.8 1 . 3 )
0.8 (0.7 1.2)
0.4 (0.3-0.5) 0.5 (0.3-0.8) 0.6 (0.3-0.7)
20:2
1.1 (0.8-1.4)
0.6 (0.4-0.7)
0.3 (0.2 0.4)
20:3
1.7 (1.2-2.3)
6.5 (5.0-7.8)
5.6 (4.5 6.7)
1.0 (0.8 1.3) 0.9 (0.6-1.1) 2.0 (1.5-2.5)
0.9 (0.6-1.4)
20:4
Values are the mean of five horses (range observedt and are expressed as per cent of total fatty acids. Horse Nos 1 5 were used. Abbreviations: CE, cholesterol ester: PC, phosphatidylcholine: PE, phosphatidylethanolamine: LPC, lysophosphatidylcholine and TG, triglyceride.
1.0 (0.8-1.4) TG 2.0 (1.7-2.4)
1.4 (0.8-1.8) LPC
PE
2-position
l-position
0.7 (0.4-0.8) PC Total
CE
14:0
Table 3. Fatty acid composition of serum lipids
O
3
8
e"
,--i
188
MUNEH1KO YAMAMOTO, YUKIO TANAKA and MICHIHIRO SU(;ANO o 3L
~..,~ ~
CE LPC
6c
--~ o E
02 m 4c ,,= ol
/
FFA
::L .
~
0
x TC and TPL
3C
~' TG E
7o E ~
/
~ 5c N.
-- 80
,(3
-01
e -
-o3
o
/
t",,
I
0
1 2
I 4
--~ 2C o
/
/
/
/
/-
6o~
/,"
so gS
/
o+-
- 3 o g_~
Y
/
.¢.7. o
Z
_ 20 J3 '~_
~___.
P I~D- I ~ , , o m ii
J I 6 i2 Incubation time,
2
PC I FC 24 hr
Fig. 1. Time course of changes in the concentration of serum lipids during incubation, Values are the mean of five horses (Nos 1-5). Abbreviations: CE, cholesterol ester: FC, free cholesterol: TC, total cholesterol: FFA, free fatty acids: TG, triglyceride: TPL, total phospholipids: PC, phosphatidylcholine and LPC, lysophosphatidylcholine.
4
6
,: 12 Incubation time,
to c~ .E
24 hr
Fig. 3. Effect of incubation on the incorporation of 4-~4C cholesterol in Tween 20 into cholesterol esters and percentage distribution of radioactivity among individual esters. Values are the mean of three horses (Nos. 3 5). © O. total incorporation: • -O, saturated esters: • •. monoenoic: [] C], dienoic and U II, tetraenoic.
Stoichiometry of cholesterol ester!tication of lysolecithin during incubation are shown in Fig. 2. The proportion of stearic acid increased, while that of palmitic acid appeared to decrease slightly. Oleic, linoleic and arachidonic acids decreased to a lesser extent. Consequently, the fatty acid composition of lysolecithin after long time incubation resembled that of the l-position of lecithin (Table 3). Ester!fication qf 4J4C-cholesterol The results are given in Fig. 3. The rate of esterification of 14C-cholesterol in Tween 20 proceeded
linearly at least for 2 hr, in agreement with that demonstrated by chemical analysis (Fig. l). The distribution of radioactivity among individual cholesterol esters was constant irrespective of incubation periods and was similar to the fatty acid composition of that fraction (Table 3).
As shown in Fig. 4, the increase in m o n o u n s a t u r ated (mainly oleic acid) and diunsaturated (mainly linoleic acid) cholesterol esters during incubation was quantitatively compensated by the decrease in the corresponding acids of the 2-position of lecithin. In contrast, the increase in saturated fatty acids (mainly palmitic acid) in cholesterol ester was more t h a n twice the decrease in lecithin.
Rerersible inhibition qf the L C A T reaction by a SH blocking reagent The esterification was completely inhibited by an addition of 10 m M 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (Tokyo Chemical Industry Co.. Tokyo). The
C E - di ene.
02-zL
60--
*~ 50 ~
18:0
f :E_-a0 c
"o --
~
C E - said.
"5
~
C E - mono.
o
'
o
o
-~'
~
16:0
i§_0.,,
h
•
~
• P C - satd. • P C - mono.
--
\,
20--
._~ ~-02 '
2
4
'
6 12 Incubation time,
.
18:1
e
18:2
'~ 2 0 : 4 24 hr
Fig. 2. Time course of changes in the fatty acid composition of lysophosphatidylcholine. Values are the mean of five horses (Nos 1-5). Carbon chain length:numbers of double bonds.
o
•
I 2
L 4
I L 6 12 Incubation time,
PC-diene.
i 24 hr
Fig. 4. Quantitative analyses of fatty acids in cholesterol ester and phosphatidylcholine. Values are the mean of five horses (Nos 1 5). Satd, saturated fatty acids: Mona, monounsaturated and Diene, diunsaturated.
Serum and liver lipid composition in horses
189
higher t h a n that demonstrated in h u m a n and rat by the above authors.
40
Fatty acid specificity of cholesterol esterification ;7-
2o
0
I
2 3 4 Incubation
5 6 time,
9 hr
Fig. 5. Effect of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) on the LCAT activity and the reactivation by 2-mercaptoethanol. Serum (0.3ml) was incubated with 0.03 ml of a Tween 20 solution containing 0.06/~Ci 4-~4C cholesterol and 0.03 ml of DTNB in 0.1 M potassium phosphate buffer (pH 7.1) at 37°C. After incubation for 3 hr, 0.3 ml of mercaptoethanol was then added at the concentrations shown below. O-----
