Esp. Eye Res. (1979)29, 131-140
Distribution of Arachidonic Acid and Other Fatty Acids in Glycerolipids of the Rabbit Iris*
inclusive of higher inositides, fatty acid composition, The phospholipid composition, and in vivo and in vit,ro incorporation of rW]arachidonate into the rabbit iris glycerolipids Mere determined. (1) The t&al phospholipid phosphorus was found to range betkeen 13.~14 timolle. of iris. Phosahatidvlcholine. ahosnhatidvlethanolamine. uhosnhat.idvlserine and sihing&yelin constiked lbout 8506 of the A total” lipid phosphorus. Et)hanolamine plasmalogen, choline plasmalogen and inositol-containing phospholipids constituted about 11,4 and 70; of the total lipid phosphorus respectively. (2) Analysis of the fatty acids of the various glycerolipids of the iris revealed the major saturates to be 16 : 0 and 18 : 0 and the major unsat,urates to be 18 : 1, 18 : 2 and 20 : 4. Ethanolamine-containing phospholipids contained t,he highest percentage of 20 : 4 followed by choline-containing phospholipids, diacylglycerol and phosphatidylinositol respectively. Sphingomgelin contained only trace amounts of 20 : 4. (3) It was found that the iris can incorporate, both in vivo and in vitro, [ Wlarachidonate into its own glycerolipids. In general glycerolipids isolated from the in vitro experiment contained significantly higher radioactivities than those obtained from t.he in vivo experiment. In vitro about 41!/;, of the rl*C]radioactivity was recovered in the neukal lipids and phosphatidylcholine, phoiphatidyiethanolamine and phosphatidylinositol contained onlv 10.2 and 2.89~: of the total radioactivitv resaectivelv: and in vivo about, 25?,, of the total radiokctivity w& found in each of phoschati&lcholiLe and the neutral lipi& and 8.4(:x in phosphatidylethanolamine. (4) No intraocular signs of an inflammatory response were observed 0.5, 1 and 7 hr aft,er intracameral injection of [Wlararhidonic acid (2 Fg) -bound albumin (0.1 mg). Key words: rabbit, iris; phospholipids; neut,ral lipids; fatty acids; ararhidonic acid. .L
1
”
1. Introduction Unesterified precursor fatty acids, such as arachidonic acid, are virtually undetect&le in most tissues in vivo. Thus? a controlled release from glycerolipids of the cell nlealhranes t)y acylhydrolases to t,he multienzyme complex of the prostaglandin synthetase is generally thought to i)e the rate linliting step for endogenous kiosynthesis. Although the iris can convert polyunsaturated fatty acids into prostaglandins at a much higher rate than the lens or retina (Van Dorp, Jouvenaz ant1 Struijk. 1967), there is little information concerning the distribution of arachidonic acid in the various glycerolipids of the iris. Thus, C’ulp. (~!unningham, Tucker, Jetcr and Deiterman (1970) investigated the fatty acid compostion of total phospholipitls and neutral lipids of the rabbit iris. Anderson, &laude and Feldman (1970) and C!&. Tucker. Rat13 and Hall (1970) h ave reported ou the fatty acid composition of the major phospholipids of the bovine iris. The pharmacological effects of arachidonic acid and its prostaglandin derivatires on the iris have already been reported (Eakins. I97i ; (‘rawford, Van Alphen, Cook and Lands. 19i8). As part of a st,udy on the metabolism antI function of glycerolipids in the rabbit iris (Abdel-Latif, Akhtar and Hawthornc~ ‘I 977 ; Al&+Latif, Green, Rnith, * (~‘ontribut,ion So. 515 from the Dep.Mmcnt I korgia. c,ol~---4s:~a/79/osol~l+
of C’rll antI Mr~lrrul~
Biology,
the Medical College ~,t
'(7 1979 Acatlctnis~ PUSS10~.( r~I~I1dc~ll) I,itdcd
10 $01.00~0 131
1x
A. A. AKUEL-LA’I’I
F ASI)
J. 1’. SMI’I’H
McPhmxon ad
Nlntheny. 1978; Akhtar and Abdel-Latif. 197X) we rcls~rt in t,his co~nnlurlicabio~l results from an in-depth study of: (a) The phospholipitl conq)ositiotl of the ral)l)it iris. (I)) The distribution of arachidonic acid and other fatty acids in the various glpcerolipitls of the iris. (c) The in viva and in vitro incorporatioti of [‘*C!~arachiclonatc into these glycerolipicls. 2. Materials and Methods Tissues Young New Zealand White rabbits (about 4 weeks old) of either sex, weighing approximately 600P900 g were used throughout this work. About 10 to 16 rabbits were killed by a blow to the head, the eyes were immediately enucleated, the irises removed, pooled and placed in chloroform-methanol (2 : 1) or chloroform-methanollHC1 (200 : 200 : 1). Extraction
and isolation of ylycerolipids
Lipids were extracted with chloroform-methanol-HCl and separated into the individual phospholipids by means of two dimensional TLC with silica-gel H as described previously (Abdel Latif, Akhtar and Hawthorne, 1977). In brief the total lipid extracts were combined and evaporated under N,. The lipids were then dissolved in chloroform and washed first with 0.1 M-HCl, and then three times with its “synthetic upper phase”. Sfter disca,rding the upper phase, the chloroform layer was evaporated to dryness under a stream of N, and the lipids were dissolved in chloroform, and separated into the individual phospholipids by means of two-dimensional TLC with silica-gel H. The solvent systems usetl were: chloroform-methanol-NH, (65 : 25 : 4, by vol) in the first system; and butanoll acetic acid-water (6 : 1 : 1, by vol) in the second system. The neutral lipids, which migrate with the solvent front, were scraped from the plates, eluted with chloroforn-~methauol (2 : l), then separated into diacylglycerol and triacylglycerol by means of one-dimensional TLC (Mangold and Malins, 1960). Determination
of phospholipk? conteti in the rabbit ivis
After two-dimensional TLC the phospholipids were detected by means of I, vapour, the spots were scraped from the plates and digested in 72”,/0 (w/v) HClO,. After cooling and the addition of 4 ml of water, the sample was centrifuged and the clear supernatant was analyzed for phosphate (Bartlett, 1959). Gas-liquid
chromatoqra&
(GLC)
The composition of fatty acids liberated by saponification were determined by ULC of their methylesters. A Beckman GC-65 gas chromatograph equipped with dual flame ionization detectors, a digital integrator for peak area analysis (spectraphysics) and a sta,inless steel column (6 ft x $” id.) containing 10% diethyleneglycol succinate on SO/100 mesh Supelcoport and operated isothermally (185’C) was used to separate the methylesters. Helium was the carrier gas. Identification of the glycerolipid spots was achieved by spraying with 2’,‘7’-dichlorofluorescein dye and visualization under ultraviolet light. Methylesters were prepared by methanolysis using Na-methoxide reagent (Sun and Horrocks. 1968). In brief the lipid spots (applies to both phospholipids and neutral lipids) together with silica gel are scraped (by a razor blade) into test tubes. To each tube, 1 ml of Namethoxide reagent is added and mixed thoroughly with the silica gel. Reaction is rapid and methylesters are formed instantaneously at room temperature. After the mixing, 3 ml methanol, 8 ml chloroform and 3 ml of water are added and mixed thoroughly. The flourescent dye partitions into the aqueous layer (upper phase). In order to eliminate interference by silica gel, the lower phase solvent is filtered into test tubes containing anhydrous Na,SO,. The solvent is then evaporated under N, and the samples redissolved
ARACHIDONIC
ACID
AND
GLYCEROLIPIDS
OP THE
1.‘i‘i
Distribution of Arachidonic Acid and Other Fatty Acids in Glycerolipids of the Rabbit Iris*
inclusive of higher inositides, fatty acid composition, The phospholipid composition, and in vivo and in vit,ro incorporation of rW]arachidonate into the rabbit iris glycerolipids Mere determined. (1) The t&al phospholipid phosphorus was found to range betkeen 13.~14 timolle. of iris. Phosahatidvlcholine. ahosnhatidvlethanolamine. uhosnhat.idvlserine and sihing&yelin constiked lbout 8506 of the A total” lipid phosphorus. Et)hanolamine plasmalogen, choline plasmalogen and inositol-containing phospholipids constituted about 11,4 and 70; of the total lipid phosphorus respectively. (2) Analysis of the fatty acids of the various glycerolipids of the iris revealed the major saturates to be 16 : 0 and 18 : 0 and the major unsat,urates to be 18 : 1, 18 : 2 and 20 : 4. Ethanolamine-containing phospholipids contained t,he highest percentage of 20 : 4 followed by choline-containing phospholipids, diacylglycerol and phosphatidylinositol respectively. Sphingomgelin contained only trace amounts of 20 : 4. (3) It was found that the iris can incorporate, both in vivo and in vitro, [ Wlarachidonate into its own glycerolipids. In general glycerolipids isolated from the in vitro experiment contained significantly higher radioactivities than those obtained from t.he in vivo experiment. In vitro about 41!/;, of the rl*C]radioactivity was recovered in the neukal lipids and phosphatidylcholine, phoiphatidyiethanolamine and phosphatidylinositol contained onlv 10.2 and 2.89~: of the total radioactivitv resaectivelv: and in vivo about, 25?,, of the total radiokctivity w& found in each of phoschati&lcholiLe and the neutral lipi& and 8.4(:x in phosphatidylethanolamine. (4) No intraocular signs of an inflammatory response were observed 0.5, 1 and 7 hr aft,er intracameral injection of [Wlararhidonic acid (2 Fg) -bound albumin (0.1 mg). Key words: rabbit, iris; phospholipids; neut,ral lipids; fatty acids; ararhidonic acid. .L
1
”
1. Introduction Unesterified precursor fatty acids, such as arachidonic acid, are virtually undetect&le in most tissues in vivo. Thus? a controlled release from glycerolipids of the cell nlealhranes t)y acylhydrolases to t,he multienzyme complex of the prostaglandin synthetase is generally thought to i)e the rate linliting step for endogenous kiosynthesis. Although the iris can convert polyunsaturated fatty acids into prostaglandins at a much higher rate than the lens or retina (Van Dorp, Jouvenaz ant1 Struijk. 1967), there is little information concerning the distribution of arachidonic acid in the various glycerolipids of the iris. Thus, C’ulp. (~!unningham, Tucker, Jetcr and Deiterman (1970) investigated the fatty acid compostion of total phospholipitls and neutral lipids of the rabbit iris. Anderson, &laude and Feldman (1970) and C!&. Tucker. Rat13 and Hall (1970) h ave reported ou the fatty acid composition of the major phospholipids of the bovine iris. The pharmacological effects of arachidonic acid and its prostaglandin derivatires on the iris have already been reported (Eakins. I97i ; (‘rawford, Van Alphen, Cook and Lands. 19i8). As part of a st,udy on the metabolism antI function of glycerolipids in the rabbit iris (Abdel-Latif, Akhtar and Hawthornc~ ‘I 977 ; Al&+Latif, Green, Rnith, * (~‘ontribut,ion So. 515 from the Dep.Mmcnt I korgia. c,ol~---4s:~a/79/osol~l+
of C’rll antI Mr~lrrul~
Biology,
the Medical College ~,t
'(7 1979 Acatlctnis~ PUSS10~.( r~I~I1dc~ll) I,itdcd
10 $01.00~0 131
1x
A. A. AKUEL-LA’I’I
F ASI)
J. 1’. SMI’I’H
McPhmxon ad
Nlntheny. 1978; Akhtar and Abdel-Latif. 197X) we rcls~rt in t,his co~nnlurlicabio~l results from an in-depth study of: (a) The phospholipitl conq)ositiotl of the ral)l)it iris. (I)) The distribution of arachidonic acid and other fatty acids in the various glpcerolipitls of the iris. (c) The in viva and in vitro incorporatioti of [‘*C!~arachiclonatc into these glycerolipicls. 2. Materials and Methods Tissues Young New Zealand White rabbits (about 4 weeks old) of either sex, weighing approximately 600P900 g were used throughout this work. About 10 to 16 rabbits were killed by a blow to the head, the eyes were immediately enucleated, the irises removed, pooled and placed in chloroform-methanol (2 : 1) or chloroform-methanollHC1 (200 : 200 : 1). Extraction
and isolation of ylycerolipids
Lipids were extracted with chloroform-methanol-HCl and separated into the individual phospholipids by means of two dimensional TLC with silica-gel H as described previously (Abdel Latif, Akhtar and Hawthorne, 1977). In brief the total lipid extracts were combined and evaporated under N,. The lipids were then dissolved in chloroform and washed first with 0.1 M-HCl, and then three times with its “synthetic upper phase”. Sfter disca,rding the upper phase, the chloroform layer was evaporated to dryness under a stream of N, and the lipids were dissolved in chloroform, and separated into the individual phospholipids by means of two-dimensional TLC with silica-gel H. The solvent systems usetl were: chloroform-methanol-NH, (65 : 25 : 4, by vol) in the first system; and butanoll acetic acid-water (6 : 1 : 1, by vol) in the second system. The neutral lipids, which migrate with the solvent front, were scraped from the plates, eluted with chloroforn-~methauol (2 : l), then separated into diacylglycerol and triacylglycerol by means of one-dimensional TLC (Mangold and Malins, 1960). Determination
of phospholipk? conteti in the rabbit ivis
After two-dimensional TLC the phospholipids were detected by means of I, vapour, the spots were scraped from the plates and digested in 72”,/0 (w/v) HClO,. After cooling and the addition of 4 ml of water, the sample was centrifuged and the clear supernatant was analyzed for phosphate (Bartlett, 1959). Gas-liquid
chromatoqra&
(GLC)
The composition of fatty acids liberated by saponification were determined by ULC of their methylesters. A Beckman GC-65 gas chromatograph equipped with dual flame ionization detectors, a digital integrator for peak area analysis (spectraphysics) and a sta,inless steel column (6 ft x $” id.) containing 10% diethyleneglycol succinate on SO/100 mesh Supelcoport and operated isothermally (185’C) was used to separate the methylesters. Helium was the carrier gas. Identification of the glycerolipid spots was achieved by spraying with 2’,‘7’-dichlorofluorescein dye and visualization under ultraviolet light. Methylesters were prepared by methanolysis using Na-methoxide reagent (Sun and Horrocks. 1968). In brief the lipid spots (applies to both phospholipids and neutral lipids) together with silica gel are scraped (by a razor blade) into test tubes. To each tube, 1 ml of Namethoxide reagent is added and mixed thoroughly with the silica gel. Reaction is rapid and methylesters are formed instantaneously at room temperature. After the mixing, 3 ml methanol, 8 ml chloroform and 3 ml of water are added and mixed thoroughly. The flourescent dye partitions into the aqueous layer (upper phase). In order to eliminate interference by silica gel, the lower phase solvent is filtered into test tubes containing anhydrous Na,SO,. The solvent is then evaporated under N, and the samples redissolved
ARACHIDONIC
ACID
AND
GLYCEROLIPIDS
OP THE
1.‘i‘i
