TOXICOLOGYANDAPPLIED
PHARMACOLOGY45,69-17(1978)
The Effect of Phenobarbital and Carbon Tetrachloride Fatty Acid Content and Composition of Phospholipids Rat Liver M. S.
ILYAS,
F. A.
DE
LA
IGLESIA,
AND
G.
Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, LambertfParke-Davis Pharmaceutical Research Division, Ann Arbor, ReceivedJuly
19,1977;
accepted
November
on from
FEUER Canada Michigan
and Warner48106
15, I977
The Effect of Phenobarbital and Carbon Tetrachloride on Fatty Acid Content and Composition of Phospholipids from Rat Liver. ILYAS, M. S., DE LA IGLESIA, F. A., AND FEUER, G. (1978). Toxicol. Appl. Pharmacol. 4569-77. The administration of phenobarbital or carbon tetrachloride to rats caused various changes in hepatic fatty acid content and composition. Phenobarbital elicited no effect on the total amount of fatty acids but significantly decreased myristic, pentadecanoic, and arachidonic acids and increased eicosatrienoic, eicosapentenoic, lignoceric, and docosatrienoic acid. In contrast, carbon tetrachloride enhanced significantly the total content and several components such as pentadecanoic, palmitic, palmitoleic, oleic, linoleic, arachidic, eicosenoic, eicosadienoic, eicosatrienoic, docosapentenoic, lignoceric and docosahexenoic acids. It elicited no effect on arachidonic acid. Unsaturated fatty acid moieties participating in the structure of these phosphatides were increased by phenobarbital and diminished by carbon tetrachloride. Phenobarbital caused a reduction in the ratio of saturated/unsaturated fatty acids mainly because of the decreased palmitic and increased oleic, linoleic, eicosatrienoic, arachidonic, docosapentenoic, and docosahexenoic acids. The significant variation brought about by phenobarbital and carbon tetrachloride on tissue fatty acids and in particular on fatty acid composition of phosphatidylcholine and phosphatidylethanolamine fractions reflects the opposing effects of these compounds on the liver cell. The major action of phenobarbital and carbon tetrachloride is associated with changes of the endoplasmic reticulum. Thus, their contrasting effect on fatty acid composition and metabolism may suggest that the disposition of lipid constituents plays a determinant role in the hepatic action of foreign compounds.
The close similarity in the liver response to a large number of drugs, characterized by enzyme induction and the development of a wide variety of biochemical reactions in hepatic microsomes, initiated many investigations into the mechanism of these events (Conney et al., 1960; Remmer and Merker, 1965). The lipid solubility of the inducers raised the possibility that the induction process was related to this property (Gaudette and Brodie, 1959; Gilbert and Golberg, 1967), although other experiments did not confirm this hypothesis (Feuer, 1970). It seems that the analysis of hepatocyte constituents participating in the response would offer an alternate route to investigate the basis of interaction between drugs and cellular membranes (Smuckler, 1968; Chaplin and Mannering, 1970). The lipid complement of membrane-bound enzymes constitutes an integral part of their catalytic or regulatory action (Duttera et al., 1968; Vessey and Zakim, 1971). 69
M)41-0081(/78/0451-0069%02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
70
ILYAS,
DE LA IGLESIA,
AND
FEUER
Perturbations of membrane lipids brought about by organic solvents, detergents, and phospholipase treatment altered enzyme properties, suggesting the importance of phospholipids in membrane function (Triggle, 1970; Zakim and Vessey, 1975). In particular, the synthesis of phosphatidylcholine from phosphatidylethanolamine and the breakdown to lysophosphatidylcholine were altered by various drugs. Modification of these phospholipid components in the endoplasmic reticulum membrane by inducers resulted in activation, whereas hepatotoxic compounds elicted inhibition (Feuer et al., 1972, 1974). Phospholipid biosynthesis and methyl transfer reactions in hepatic microsomes are intrinsically associated with drug actions (Feuer et al., 1973; AcheampongMensah and Feuer, 1975). Two possibilities can be raised for explaining the interaction between chemicals and lipids on the function of the endoplasmic reticulum. The effects may occur because (1) enzymes which are responsible for the synthesis and distribution of membrane-bound lipids are influenced directly by these chemicals; or (2) enzymes are altered indirectly by some primary effects of chemicals on the distribution of membrane-bound lipids. In either case treatment-related alterations may manifest in differences in lipid composition of the endoplasmic reticulum which may be extended to other cellular components. To address ourselves to these questions, the effects of phenobarbital (PB), as a representative inducer, and carbon tetrachloride (Ccl,) as a representative hepatotoxin, on fatty acid composition of rat liver and hepatic endoplasmic reticulum have been investigated. In this paper the effect of these chemicals on total hepatic fatty acids is reported. The action on the endoplasmic reticulum of liver cells is reported elsewhere (Ilyas et al., 1978). METHODS Animals. Male albino Wistar rats (Woodlyn Farm, Guelph, Ontario) weighing 150 to 200 g were housed in individual cages and maintained on laboratory chow (Purina Laboratory Chow, Ralston Purina Co., St. Louis, Missouri) and water ad libitum. Animal quarters were well-aerated air-conditioned rooms kept at 20 rt 2OC and 50 to 60% relative ambient humidity. Housing, experimental handling, and husbandry of animals were within accepted guidelines from the Animals for Research Act of Ontario, 1971. Experimental compounds. The sodium salt of phenobarbital (0.2 mmol/kg body weight was administered in seven daily ip doses in 2.5 ml/kg saline. Ccl, (5.2 mmol/kg) was given in four daily ip doses in 2.5 ml of arachis oil/kg. The last injections of PB and Ccl, were given 4 and 18 hr before sacrifice, respectively. The number of animals in each group was six, and controls received the equivalent volume of vehicle. Preparation of liver homogenate. Rats were killed under light anesthesia by exsanguination from the vena cava. The liver was rapidly removed, rinsed with ice-cold physiological saline solution, and homogenized in 1.15% KC1 solution in 0.1 M Tris buffer (PH 7.4) so that 3 vol of medium was added to each gram of tissue. Lipid extraction. The glassware and other items used for this procedure were thoroughly cleaned and made fat free by washing with a hot chloroform-methanol mixture 12: 1 (v/v)]. The determination of total fatty acid content was carried out in lyophilized homogenates. To the lyophilized residue was added 10 ml of methanol, and they were mixed and left standing for 4 to 8 hr in the dark at room temperature. The
EFFECT
OF
PB
AND
ccl,
ON
HEPATIC
FATTY
ACIDS
71
content was then mixed with 20 ml of chloroform and allowed to stand for 2 hr at room temperature. The suspension was filtered through defatted Whatman filter paper and the sediment was washed twice with 10 ml of chloroform-methanol mixture (2: 1). The combined filtrate and washings were evaporated to dryness in a rotary evaporator at 37°C and the dry residue was transferred to a screw-cap tube. Isolation of individual phospholipids. The phospholipid fraction was extracted from 0.5 ml of 25% liver homogenate and purified according to the method of Folch et al. (1957), as modified in our laboratory (Cooper and Feuer, 1972). The extracts were evaporated to dryness and transferred quantitatively into lo-ml volumetric flasks. The entire extract was then reduced to a OS-ml volume and quantitatively spotted onto silica gel G coated glass plates. Phosphatidylcholine and phosphatidylethanolamine were separated by thin-layer chromatography using a solvent system of chloroform/methanol/water (65 :24 :4 by volume), (Skipsky et al., 1962). The phospholipid bands were located by spraying the plate with 0.5% 2,7dichlorofluorescein in 50% methanol and were visualized under ultraviolet light. Preparation offatty acid methyl esters. To the dry residue of total lipid extract was added 2.0 ml of 10% H,SO, in absolute methanol (w/v). Methanolysis was performed at 80°C for 4 hr. The tube contents were cooled and diluted with distilled water. The fatty acid methyl esters were extracted three times with 3.0-ml portions of petroleum ether. The extracts were collected in a tube and evaporated to dryness under N, gas. This residue was redissolved in 1.0 ml of petroleum ether and stored at - 18OC under N, for further gas-liquid chromatography analysis (Kuksis et al., 1968). The preparation of phosphatidylcholine and phosphatidylethanolamine fatty acid methyl esters was carried out from the spots identified on the plates. These were scraped off and methylated, as described above. Gas chromatography of fatty acids. Fatty acids were analyzed using a F and M Biochemical Gas Chromatograph Model 400, with a H, flame ionization detector. The glass columns were (3-mm id x 6 ft) packed with 10% EGSS-X on Chromosorb W, AW, 100 to 120 mesh. The gas chromatograph was operated isothermally at 180°C with N, carrier gas at a flow rate of 60 ml/min. Peak identity and quantitative validation of estimates of fatty acid concentration were done by comparison with the National Heart Institute Fatty Acid Standards (Mixtures, KA, KD, KF) and the Hormel Institute Fatty Acid Standards (Mixtures 1, 2, and 8). The quantitative data agreed with the stated composition, showing a relative error of less than 1% for major components and of less than 5% for minor components. The peak identification was further confirmed by cod liver oil fatty acid analysis (Ackman and Burgher, 1965). Calculations. Statistical analysis was carried out using the Student t test (Snedecor and Cochran, 1965) and the Dunnett test (1964) for the analysis of variance for multiple comparison with a control. The percentage recovery from gas chromatograms was computed by a rapid method (Forss, 1968). RESULTS
Phenobarbital treatment affected the level of several fatty acids in the liver and the total amount was significantly different from controls (Table 1). Myristic, pentadecanoic, and arachidonic acid content was reduced and eicosatrienoic, eicosapen-
12
ILYAS,
DE LA IGLESIA,
TABLE FATTY
ACID
COMPOSITION
OF THE WHOLE
14:ob 15:o 16:0 16: 1 18:0 18:l 18:2 20:o 2O:l 20:2 20:3 20~4 20:5 22~3 22~5 22:6 24:0 Saturated Unsaturated Total
347.00
f
6.51
2.65
6.27
1.25 18.18 1.65 16.86 12.08 19.53
144.44 + 2.59c 2,321.43 & 39.30 218.49 + 4.88
0.62 0.36 0.77 0.87
83.11 + 1.68 46.22 f 0.91 101.11 + 2.24 150.45 + 4.25c 1,022.44 z! 24.5 lc 281.00 + 5.69c 155.33 + 3.98’
?r 21.80
215.87 2,212.44 1,584.33 2,561.67 81.33
2 f + k +
6.08 34.58 24.89 16.88 2.03
k
0.69
47.52
100.67 113.62 1,837.33 164.11 127.44 321.67
?r 2.27 i 1.52 + 25.37 f 5.48 + 1.21 f 3.81
14.01 1.15 0.97
f
5.39
107.78 f
3.66
2.45 5.87 0.82
5,298.11 f 41.14
40.39
7,820.70
59.80
745.89
+ 42.85
13,118.81 f 50.70
IN RATP
100.19
234.22
2,255.89
it
8.44'
+ 26.04
1,597.33 + 21.27 2,536.89
326.78
+ 27.04
;
CCI, %
MfP
164.45 f 2,385.22
LIVER
PB %
iudg
FEUER
1
Control Fatty acid
AND
3.60
1.88 1.17 18.73 1.76 18.20 12.89 20.46 0.67 0.37 0.82
1.21 8.25 2.27
1.25 2.64
757.11 + 12.87 164.22 + 3.52’
6.10
5,201.78 + 49.02 7,193.11 + 45.66’
41.97
12,394.89
+ 79.84'
0.32 57.88 99.85
%
PdP 6.99 7.36=
1.85 1.03
4,413.33 + 69.68’ 457.11 It: 6.75c
22.47 2.32
2,260.78 3JN7.55 4,375.55
11.51 15.52
363.33 202.33
+_ f
+
30.63
f
41.7gc
f
64.43' 3.05c
169.33 + 78.33
218.05 159.33 1,873.22 163.00 129.78 441.00 1,120.56 163.05
k
f + i + * + i -t
1.71c 11.29’ 5.5w 25.12 2.13 1.67 11.36’ 33.93’ 7.40c
7,571.89 + 81.94c 12Jh53.67 f 91.96c 19,635.56 f 162.88~
22.28 0.86 0.39
1.11 0.81 9.53 0.83 0.66 2.24 5.70 0.83 38.56
61.38 99.94
u Values are expressed in micrograms of fatty acid per gram of liver and given as the mean _+ SE; % indicates the percentage value of the total fraction. QNumbers represent the total carbon atoms and double bonds in the individual fatty acid moiety. c Indicates significant differences from controls, p < 0.05.
tenoic, docosatrienoic, and lignoceric acids were increased. In contrast, administering Ccl, caused a significant rise of total hepatic fatty acids. The increases by Ccl, were seen in the levels of pentadecanoic, palmitic, palmitoleic, oleic, linoleic, arachidic, eicosenoic, eicosadienoic, eicosatrienoic, docosapentenoic, docosahexenoic, and lignoceric acid. Ccl, elicited no effect on arachidonic acid content. Some individual fatty acids such as pentadecanoic acid showed opposite changes, with increases because of Ccl, and decreases with PB. There was no change in the level of docosahexenoic acid by the administration of PB, but the level was significantly increased by Ccl,. No change was seen in the stearic acid fraction by either treatment. The effect of PB and Ccl, modified the percentage distribution of hepatic fatty acids. The ratio between saturated and unsaturated fatty acids was altered from 40/60 in controls to 42/58 with PB and 38/62 with Ccl,. Fatty acids in the phosphatidylcholine fractions represented 18, 20, and 10% of the total fatty acids in the liver of control, PB, and Ccl,-treated rats, respectively. This represented a 7% increase with PB and a statistically significant reduction of 12% with Ccl,. Several fatty acids present in liver were not detected in the phosphatidylcholine fractions. Moreover, differences were found in the distribution of individual fatty acids between total tissue and phosphatidylcholine fractions (Table 2). Phenobarbital treatment significantly increased myristic, pentadecanoic, stearic, eicosatrienoic, and eicosapentenoic acids and reduced palmitic acid. Ccl, administration significantly
EFFECT
OF
PB
AND
ccl,
ON
HEPATIC
TABLE FATTY
ACID COMPOSITION
OF LIVER
FATTY
73
ACIDS
2
PHOSPHATID~LCHOLINE
FRACTIONS
IN RATS’
Treatment PB
Control Fatty acid 14:Ob 15:o 16:0 16:l 18:O 18: 1
18:2 2O:l 20:2 20:3 20~4 20:5 22:5 22~6
Saturated Unsaturated Total
%
Pdg 4.02 7.32 556.39 36.85 427.83 213.62 347.79 9.01 4.78 13.12 457.57 68.66 18.08 167,85
+ 0.19 k + f k & * + f f + + + 2
%
Kdg
0.17
0.43 22.13 3.72 13.46 16.82 13.54 0.64 0.31 0.58 21.05 3.81 1.04 7.54
0.31 23.84 1.58 18.34 9.15 14.91 0.38 0.20 0.56 19.61 2.94 0.77 7.19
995.57 f 28.66 1337.35 f- 58.57 2332.92 k 82.08
42.67 57.28 99.95
8.12 11.42 411.39 40.42 558.74 241.00 383.20 9.06 5.75 15.42 487.34 140.28 19.50 169.28
& + t f k f f. + + f k + 5 +
ccl,
0.24c 0.58c 21.55c 4.09 23.35’ 15.15 9.27 0.74 0.50 1.26c 36.44 11.08c 2.04 8.46
989.58 _+ 25.09
0.32 0.45 16.44 1.61 22.34 9.63 15.32 0.36 0.23 0.61 19.48 5.60 0.78 6.77 39.57
1511.26f 51.59 60.40 2500.84 + 58.50
99.97
dg 3.84 8.12 528.50 31.78 448.39 189.03 284.00 7.47 4.74 10.36 347.70 63.66 11.93 141.79
+ 0.33 f 0.40 f 27.75 + 2.33 f 23.91 f 13.25 & 16.74c k 0.25 &- 0.42 & 0.77c f 25.44c + 8.46 + 0.50c + 7.41c
% 0.18 0.39 25.63 1.54 21.75 8.19 13.77 0.36 0.23 0.50 16.86 3.08 0.58 6.88
988.76k 50.00 47.96 1072.84 k 37.84c 52.02 2061.60 &- 53.96c 99.98
0Valuesare expressed in micrograms of fatty acidper gramof liver and givenasmeanf SE; indicates thepercentage valueof thetotalfraction. bNumbers represent thetotalcarbonatomsanddoublebondsintheindividualfatty acidmoiety. cIndicatessignificant differences fromcontrols,p < 0.05.
%
decreased linoleic, eicosatrienoic, arachidonic, docosapentenoic, and docosahexenoic acids. The saturated/unsaturated fatty acid ratio was also modified by the experimental treatments, although to a small extent (control, 43/57; PB, 40/60; and Ccl,, 48/52). Fatty acids in the phosphatidylethanolamine fractions represented9, 11, and 6% of the total hepatic fatty acids of control rats and rats receiving PB or Ccl,, respectively (Tables 1 and 3). This resulted in a net 21% increaseby PB and a 3% decreaseby Ccl,. The fatty acid composition of these fractions was different from whole liver and phosphatidylcholine fractions to the extent that many acids were not detectable. Phenobarbital significantly enhanced the concentration of unsaturated fatty acids such as palmitoleic, oleic, linoleic, eicosatrienoic, arachidonic, docosapentenoic, and docosahexenoic acids. In contrast, Ccl, reduced all of these except stearic and oleic acids, whereaspalmitic acid content was significantly raised. Thus, the saturated/unsaturated fatty acid ratio was altered by PB (42/58) and Ccl, (54/46) from control values (47/53). DISCUSSION
There is evidence that various foreign compounds modify the phospholipid content of hepatic microsomesin the rat. Drugs which induce drug metabolism causean increase
74
ILYAS,
DE
LA
IGLESIA,
AND
TABLE FATTY
ACID
COMPOSITION
FEUER
3
OF LIVER-PHOSPHATIDYLETHANOLAMINE
FRACTIONS
IN RATSO
Treatment Control Fatty acid 16:ob 16:l 18:O 18:l 18:2 20:3 20:4 22:5 22~6
Saturated Unsaturated Total
Pugk 246.00 + 7.21
PB
%
PdP
+ 12.53 k 0.55c i 9.32 + 2.80c k 5.54c + 0.74c 325.79 + 8.75c 15.40 + 0.33' 128.95 + 3.32c 539.17 * 14.31 47.45 574.00 k 14.61 597.04 f 12.99 52.52 795.24 f 12.93c 1136.21 t 25.73 99.97 1369.24 + 26.39’ 9.94 293.17 130.40 95.60 6.88 251.16 11.67 91.39
& rf & & + & k +
0.76 7.94 8.97 5.57 0.28 4.35 0.76 5.60
21.65 0.87 25.80 11.47 8.41 0.60 22.10 1.03 8.40
255.67 12.42 318.33 165.66 129.80 17.26
ccl,
% 18.67 0.91 23.24 12.10
%
&Ii%
1.26 23.79 1.12 9.42
283.33 6.74 316.83 118.33 77.50 3.36 217.53 9.65 72.03
41.92 58.04
600.17 k 16.75 505.12 + 8.05
9.48
+ f k & f & k & &
8.67c 0.4gc 9.06 3.11 3.19c 0.26c 7.41c 0.32' 3.50'
25.63 0.61 28.66 10.70 7.01 0.30 19.68 0.87 6.51 54.30 45.68
99.96 1105.29 ? 10.86 99.98
0 Values are expressed in micrograms of fatty acid per gram of liver and given as mean i SE; % indicates the percentage value of the total fraction. b Numbers represent the total carbon atoms and double bonds in the individual fatty acid moiety. c Indicates significant differences from controls, P < 0.05.
in microsom_al phospholipids, whereas hepatotoxins bring about a reduction (Feuer et al., 1972, 1974). The difference between these effects is associated with changes in the biosynthesis of phosphatidylcholine from phosphatidylethanolamine by stepwise methylation and in lysophosphatidylcholine production (Smuckler, 1968; Chaplin and Mannering, 1970). The incorporation of [Me-‘4Clmethionine into phosphatidylcholine is greater in microsomes from rats treated with PB or other drugs, whereas this is lower with the administration of Ccl, or other toxic compounds (Cooper and Feuer, 1972). These opposite actions appear to be related to the activity of S-adenosylmethionine: microsomal phosphatidylethanolamine methyl transferase (Feuer et al., 1974). Although the effects of drugs have been identified by modifying phospholipid bases, some experiments revealed the biological importance of modifications of the fatty acid side chain. Incorporation of fatty acids into the glycerol moiety is an essential process in the biosynthesis of hepatic phospholipids (Kennedy, 1961). Diet-induced differences have been reported in the composition of rat liver phosphatidylcholine in terms of molecular species (Van Golde et al., 1965, 1968); structural and metabolic interrelationships have been revealed (Holub and Kuksis, 197 l), and drug actions on fatty acid composition of hepatic phospholipids have been demonstrated (Ariyoshi and Takabatake, 1972a,b). Enzymes involved in some biosynthetic mechanisms show specificity for the utilization of fatty acid precursors (Lands and Hart, 1966). The breakdown of phosphatidylcholine into lysophosphatidylcholine in the liver is normally catalyzed by phospholipase A, although the specificity of this catabolic enzyme in the liver has not been established. Nevertheless the metabolic rate by snake venom phospholipase A affecting
EFFECT
OF
PB
AND
ccl,
ON
HEPATIC
FATTY
ACIDS
15
various lecithins in vitro showed marked differences (Triggle, 1970; Zakim and Vessey, 1975). The results of our studies revealed significant effects on hepatic fatty acid composition between PB and Ccl,. Administration of PB decreased the quantity of total fatty acids in the liver. This treatment caused a shift toward more saturated than unsaturated fatty acids, in particular, the formation of arachidonic acid was significantly reduced. Following Ccl, treatment, the total content of fatty acids was enhanced, particularly that of saturated fatty acids. The increased fatty acid levels caused by Ccl, have been well documented (Recknagel, 1967). The changes in total fatty acid content of liver by Ccl, and the shifts in saturated/unsaturated fatty acid ratio by PB and Ccl, may be due to alterations in the synthetic rates or can be attributed to increased storage or reduced catabolism and transport. Further results from our laboratory on the action of these test compounds have shown that PB significantly raised and Ccl, reduced total fatty acid content and that these actions were associated with changes in the fatty acid composition of hepatic endoplasmic reticulum membranes. (Ilyas et al., 1978.) These findings may suggest that the contrasting action of the chemicals are probably related to changes in synthesis. In contrast to the effect of PB on the total hepatic level of fatty acids, PB increased the amount of fatty acid bound to phosphatidylethanolamine, whereas Ccl, resulted in a significant reduction in fatty acids bound to phosphatidylcholine. These results correlated with the PB elevation and the decrease by Ccl, in the amount of phospholipids (Cooper and Feuer, 1972). The administration of PB and Ccl, elicited contrasting effects on the saturated/unsaturated fatty acid ratio in the phosphatidylcholine and phosphatidylethanolamine fractions. Phenobarbital shifted the distribution of fatty acid to raise the unsaturated fatty acids, whereas Ccl, reduced unsaturated and increased saturated fatty acids. In adequate concentrations, PB and some drugs stimulate proliferation and protein and phospholipid synthesis of the hepatic endoplasmic reticulum (Conney et al., 1960; Remmer and Merker, 1965; Feuer et al., 1972). In contrast, Ccl, and other toxic compounds cause fragmentation of membranes and inhibition of protein and phospholipid synthesis (Recknagel, 1967; Smuckler, 1968; Feuer et al., 1974). There is a lack of precise knowledge concerning the primary events which determine whether the response of the liver cell is a protective proliferation or represents the initiation of a deleterious effect. The manifestation of these actions occurs at the subcellular level, and the target organelle is the endoplasmic reticulum. Although the present report is concerned with the overall hepatic effects of an inducer and a hepatotoxin only, their action on the content and composition of hepatic fatty acids attached to the phosphatidylcholine and phosphatidylethanolamine moieties revealed contrasting effects which could represent drug or toxic action. These investigations, however, should be pursued further to provide more information on the basic events leading to the development of the induction process as well as to analyse the biochemical sequences preceding pathological manifestations and final derangement of cell function. ACKNOWLEDGMENTS This work was supported by the Medical Research Council, to whom our thanks are due. We are indebted to Mr. L. Marai for the valuable help in the gas chromatographic analysis.
76
ILYAS,
DE LA
IGLESIA,
AND
FEUER
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EFFECT OF PB AND Ccl,
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