253
Effects of Aging on the Composition and Metabolism of Docosahexaenoate-Containing Lipids of Retina N.P. Rotstein, M.G. llincheta de Boschero, N.M. Giusto and M.I. Aveldafio* Instituto de Investigaciones Bioqui'micas,Universidad Nacional del Sur and Consejo Nacional de Investigaciones Cientfficas y T@cnicas, 8 0 0 0 Bahia Blanca, Argentina The amount of doeosahexaenoate (22:6n-3)-eontaining phospholipid species decreases with aging in the rat retina. Most lipids, but especially choline and serine glyeerophospholipids, show a significant fall in 22:6n-3, which is not compensated by increases in other polyenoic fatty acids. The decrease not only affects 22:6 but also various very long chain n-3 hexaenoic fatty acids which, in phosphatidylcholine, have up to 36 carbon atoms, and which are probably synthesized by successive elongations of 22:6n-3. The in vitro incorporation of [2-3H]glycerol into retinal lipids indicates that the de novo biosynthetic pathways are not impaired by aging. The incorporation of [1-14C]docosahexaenoate is significantly stimulated into all lipids of aged retinas, but to the largest extent in those showing the largest decreases in 22:6, especially in choline glycerophospholipids. The results indicate that the decreased levels of 22:6 with aging are due not to an impaired activity of the enzymes involved in the synthesis and turnover of phospholipids but to a decreased availability of this polyene in the retina. It is suggested that this may stem from a defect in some of the enzymatic steps that lead to the synthesis of 22:6n-3, probably that catalyzed by A4desaturase, the effect on longer hexaenes being secondary to the decreased synthesis of 22:6. Lipids 22, 253-260 (1987). Alterations of the ratios between phospholipid headgroup classes as well as in the quality and proportions of their fatty acyl moieties are known to bring about dramatic changes in the physicochemical properties of biological membranes, along with many of their functions (1). Qualitative or quantitative changes of this type, affecting lipids, may play an important role among the biochemical causes of the insidious deterioration that many cell functions undergo during the process of aging, since lipids make the matrix where a variety of enzymes, receptors and transport systems work. Polyunsaturated fatty acid-containing lipids are especially likely to be involved, since the synthesis of their polar and nonpolar moieties-particularly that of long chain polyenoic fatty acids-needs the concurrence of, and delicate equilibrium between, many enzymatic activities. The retina is an excellent model to study these problems in mammals because its lipids are characterized by a high proportion of such polyunsaturates. The most abundant fatty acids in visual cells of vertebrates are polyenes of the n-3 series, the major representative being 4,7,10,13,16,19-docosahexaenoate (22:6n-3). The retina also contains a whole variety of tetra-, penta- and hexaenoic fatty acids along with 22:6. These include familiar polyenes like 20:4n-6 or 22:5n-3 and various polyenes whose chain lengths range from 24 to 36 carbon atoms (2,3). The latter are highly concentrated in dipolyunsaturated molecular species of *To whomcorrespondenceshould be addressed at Instituto de InvestigacionesBioquimicas,Gorriti43, 8000 BahiaBlanca,Argentina~
phospholipids of photoreceptor membranes, specifically in phosphatidylcholine (PC) (2). This paper is concerned with the effects of aging on compositional and metabolic aspects of retina phospholipids. It is shown that the levels of docosahexaenoate as well as of other n-3 hexaenes are decreased in aged retina glycerophospholipids, especially in those of choline and serine. The in vitro labeling of retina lipids with [2-3H]glycerol and [1-~C]docosahexaenoate is also compared in young and aged animals. The incorporation of this polyenoic fatty acid is shown to be markedly stimulated with aging in most retina lipid classes, but especially in those showing the largest decreases in 22:6 levels.
MATERIALS AND METHODS Wistar rats kept under constant environmental conditions and fed for various generations the same pelleted standard chow diet were used for the present experiments. No sex-related differences were found in the effect of aging on lipids. Rats aged 2-3 and 26-27 mo were killed by decapitation, their eyes were rapidly enucleated, and their retinas were dissected under a magnifying glass. Some of the retinas were immediately homogenized with chloroform]methanol and were destined to lipid and fatty acid composition analysis. Others were incubated with [2-3H]glycerol and 4,7,10,13,16,19-[1-~4C]docosahexaenoic acid (['4C]22:6) to determine the labeling of their lipids with these precursors. For this purpose, the retinas were preincubated for 5 min at 37 C in 0.8 ml of a bicarbonate~ based ionic medium, pH 7.4, containing 2 mg/ml of glucose (4), after which 0.2 ml of the same medium containing the precursors (5 nmo1114C]22:6 and 0.16 nmol [~H]glycerol) was added, and incubations (37 C) proceeded for the periods indicated in Results. At the end of incubations, 9 ml of ice-cold medium was added and the tubes containing the retinas were centrifuged (10 min at 10,000 rpm). After supernatants were discarded, the pellets were extracted with chloroform]methanol. Protein was determined according to the method of Lowry et al. (5).
The extracts from labeled and unlabeled samples were partitioned and washed according to Folch et al. (6), and the lipids were resolved by means of thin layer chromatography (TLC) (7). After development, the lipid spots were located by exposing the plates to I2 vapors and subjected to phosphorus analysis (7). The spots containing [3H]- and ['4C]-labeled lipids were scraped from TLC plates and transferred to vials containing 0.4 ml water. Ten ml of a solution containing 0.4% Omnifluor (New England Nuclear, Boston, Massachusetts) and 20% Triton X-100 in toluene was then added, and radioactivity was measured by liquid scintillation counting. The [2-3H]glycerol used (sp act 10 Ci/mmol) was from New England Nuclear, and the (1-'4C]docosahexaenoate (sp act 40 ~Ci/~mol) was provided by H. Sprecher, Ohio State University {Columbus, Ohio). LIPIDS, Vol. 22, No, 4 (1987)
254
N.P. ROTSTEIN ET AL. For f a t t y acid analyses, the lipids were isolated as before, the spots were located under UV light after spraying with dichlorofluorescein, and m e t h y l esters were p r e p a r e d {under N2} b y BF3-catalyzed m e t h a n o l y s i s {8}. The latter were analyzed b y gas liquid c h r o m a t o g r a p h y (GLC). Two glass columns (2 m • 2 m m i d } packed with 15% OV-275 on 80-120 Chromosorb W A W (Varian, Sunnyvale, California} were connected to two flame ionization detectors operated in the dual-differential mode. A linear (5 C/min) t e m p e r a t u r e p r o g r a m , s t a r t i n g at 160 C and ending at 220 C, was used, and the injector and detector t e m p e r a t u r e s were 220 C and 230 C, respectively. To obtain the d a t a in Table 3, after reaching 220 C the oven t e m p e r a t u r e was held c o n s t a n t at t h a t value for 45 min to permit the analysis of v e r y long chain polyenoic f a t t y acids (2}. RESULTS
Effects of aging on retina phospholipids. The concentration of phospholipids, expressed as the a m o u n t of lipid p h o s p h o r u s per m g of retina protein, was lower b y a factor of a b o u t 20% in retinas f r o m aged r a t s t h a n in those f r o m y o u n g animals {Table 1). Such a difference in t o t a l lipid content was not significant statistically, as when the levels of individual phospholipids were considered. The average a m o u n t s of protein were 0.70 +_ 0.11 and 0.64 +_ 0.11 m g per retina in samples f r o m y o u n g and aged retinas, respectively, while those of t o t a l p h o s p h o r u s in lipid e x t r a c t s from the s a m e samples were 250 + 34 and 189 ___ 25 nmol, respectively. While the average decrease of 8% in protein was not statistically significant, t h a t of 24% in lipid p h o s p h o r u s was P < 0.05. Hence, the cont e n t s expressed as lipid/protein ratios in Table 1 show no large differences in lipid concentration because b o t h p r o tein and lipid are affected b y aging, b u t it is a p p a r e n t f r o m the above results t h a t lipids tend to be decreased relatively more t h a n proteins. When the phospholipid composition {%) was analyzed, again no significant differences due to aging were readily a p p a r e n t {Table 1).
However, some interesting trends were observed by calculating the ratios between individual phospholipids. Most of the age-related differences in total phospholipid concentration were contributed by decreases in the three major phospholipids: phosphatidylcholine, ethanolamine glycerophospholipids (EGP) and phosphatidylserine (PS), which resulted in changes of certain phospholipid ratios. A m o n g choline phospholipids, for instance, the amount of P C was decreased to a larger extent than that of sphingomyelin (Sph, unchanged}, giving rise to a significantly lower PC/Sph ratio in aged retinas. A m o n g acidic phospholipids, PS was more decreased than phosphatidylinositol {PI}, which also resulted in a significant difference in the P S / P I ratio with age. The PC/PS ratio was similar in control and aged retinas, indicating t h a t both were diminished to the same extent, and the PC/EGP ratio was increased. The f a t t y acid composition of retina glycerc~ phospholipids was next examined in search for the possible i n v o l v e m e n t of polyenoic f a t t y acids in these effects, since it was a p p a r e n t t h a t the largest decreases affected those lipids t h a t contribute the largest a m o u n t s of the m o s t highly u n s a t u r a t e d f a t t y acids to retinal membranes. Fatty acids of retina glycerophospholipids. The m o s t conspicuous effect of aging on the f a t t y acid composition of lipids was a general tendency to a decreased proportion of 22:6n-3 {Table 2}. I t s percentage was a b o u t 30%, 10% and 13% lower in PC, E G P and PS, respectively, in aged t h a n in y o u n g animals. If one considers t h a t the amounts of these phospholipids were also lower {Table 1), it m a y be e s t i m a t e d t h a t the a m o u n t of 22:6 per m g of protein decreased b y a b o u t 42%, 31% and 30% as a consequence of the changes in such lipids {Table 4}. I t is evident f r o m Tables 2-4 t h a t no f a t t y acid was synthesized in retina to compensate for this decrease in 22:6, the slight increases observed in the percentages of some f a t t y acids being only relative to this diminution. The average unsaturation of lipids tended to decrease (Table 2}, the m o s t significant effect {P < 0.05} being observed for PC and PS. The f a t t y acid composition of PC is expanded in Table 3
TABLE 1 Content and Composition of Phospholipids in Rat Retina
Controls PC EGP PS PI PA Sph DPG Total phospholipid PC/EGP PS/PI PC/Sph
315 • 47
32 • 7
=
159.1 + 21.8 {45.7 • 115.1 • 15.2 {33.1 • 40.8 • 6.1 (11.6 + 16.7 • 3.3 {4.8 • 5.6 • 1.5 (1.6 • 5.7 • 1.9 {1.6 • 4.6 _+ 3.0 {1.3 • 350.0 • 52.0 1.38 • 0.04 2.46 • 0.20 29.40 • 6.50
Aged 0.9} 0.7} 0.5} 0.2} 0.2) 0.2} 0.5)
254 • 39
34 •
6
131.7 89.1 32.8 16.2 5.2 7.8 5.3 289.0 1.48 2.01 16.95
+ 20.2 {45.3 _+ 0.6} _+ 14.3 (31.6 -I-_1.4} • 3.9 (11.3 • 0.4} • 0.9 (5.7 • 0.5} • 1.6 {1.6 • 0.4) _+ 0.6 {2.6 • 0.4} • 4.0 {1.7 • 0.7) • 47.0 • 0.02* +- 0.20* -+ 2.24*
Controls/aged 0.83 0.77 0.80 0.97 0.91 1.37 1.15
PC, phosphatidylcholine;EGP, ethanolamine glycerophospholipids;PS, phosphatidylserine;PI, phosphatidylinositol;PA, phosphatidic acid;Sph, sphingomyelin;DPG, diphosphatidylglycerol.The figuresdepictthe amount of phospholipidas nmol/mg protein.The percentage composition is given in parentheses.Both are presented as mean values • S.D. from 4 samples (each containing6-8 retinas}from control(2.5too)and aged {26.5too)rats.Lipidswere separatedby tw~dirnensionalthinlayerchromatography. The ratiosbetween lipids were calculatedfor individualsamples and then averaged. *, Significantdifferenceswith respect to controls (P < 0.05 or lower). LIPIDS, Vol. 22, No. 4 (1987)
255 AGING AND R E T I N A D O C O S A H E X A E N O A T E
TABLE 2
Fatty Acid Composition (Mol%) of Rat Retina Glycerophospholipids
Phosphatidylcholine
Ethanolamine glycerophospholipids
Phosphatidylserine
Phosphatidylinositol
2.5 mo
26.5 mo
2.5 mo
2.5 mo
2.5 mo
0.4 + 0.1 0.6 • 0.1 35.7 +_ 1.0 1.6 • 0.1 18.3 • 1.6 22.2 • 1.5" 1.1 +_ 0.6 0.3 • 0.01
0.7 + 0.8 0.9 • 1.0 6.8 • 1.0 -30.3 • 2.8 4.8 • 0.4 0.4 + 0.1 0.1 • 0.1
14:0 15:0 16:0 17:0 18:0 18:1 18:2n-6 20:1
0.3 0.3 34.3 1.9 18.5 18.3 0.9 0.4
20:3n-6 20:4n-6 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3 24:5n-3 24:6n-3 C,~-C3~ PUFA
0.1 5.4 0.02 0.4 0.4 0.2 17.0 0.1 0.3
Average unsaturation
• 0.1 • 0.0 • 0.7 • 0.2 • 2.3 • 0.4 +_ 0.1 +- 0.1
26.5 mo
26.5 mo
26.5 mo
0.6 __ 0.6 0.1 _ 0.1 0.2 • 0.2 0.2 • 0.2 2.0 • 1.9 0.4 +_ 0.4 0.1 • 0.1 0.2 +- 0.2 -0.2 • 0.2 8.6 • 0.5 0.9 _+ 0.3 1.9 +- 1.4 8.9 • 1.5 10.7 • 3.3 -0.2 _+ 0.1 0.9 -+ 0.9 -1.1 • 0.6 32.1 • 1.3 37.2 +_ 2.9 40.8 • 2.4 35.3 • 0.6 36.0 • 1.4 5.4 • 0.5 2.1 • 0.3 3.8 • 1.0" 4.3 • 0.3 4.8 • 1.4 0.4 • 0.3 0.1 +_ 0.04 0.2 +- 0.1 0.2 _+ 0.5 0.2 • 0.2 0.1 +_ 0.03 0.1 • 0.02 0.2 • 0.2 0.1 • 0.05 0.1 • 0.1
• 0.05 0.1 • 0.05 0.2 • 0.03 0.1 • 0.04 0.1 • 0.03 0.2 • 0.2 0.1 • 0.1 0.1 • 0.1 +_ 0.6 5.7 +- 0.5 10.7 • 0.9 12.0 • 1.6 3.8 • 0.2 4.6 • 0.3 46.7 +_ 1.7 40.6 • 2.4* • 0.01 0.1 • 0.01 0.1 • 0.05 0.2 • 0.04 0,1 • 0.1 0.1 • 0.02 0.5 • 0.3 0.8 • 0.2 • 0.04 0.5 • 0.1 2.3 • 0.2 2.0 • 0.1 3.4 • 0.4 2.6 • 0.1 0.3 • 0.1 0.3 • 0.2 • 0.1 0.1 • 0.04* 1.2 • 0.2 0.4 • 0.1" 2.0 + 0.2 0.6 • 0.2* 0.3 +_ 0.2 --* • 0.03 0.3 • 0.01 0.5 • 0.1 0.7 • 0.1 1.0 +_ 0.1 1.1 • 0.2 0.1 • 0.1 +_ 1.5 11.9 • 0.9* 40.3 • 1.7 36.0 +_ 2.0* 45.3 +_ 1.8 39.3 • 2.4* 3.1 • 1.3 2.9 • 0.2 • 0.1 0.1 • 0.1 0.3 • 0.2 0.7 • 0.2 1.6 • 0.2 1.6 • 0.2 --+ 0.1 0.1 • 0.02* 0.5 • 0.2 0.4 + 0.4 2,2 • 0.3 1.6 +- 0.2* --1.2 0.7 . . . . . . . .
1.6 • 0.1
1.3 • 0.05*
3.0 • 0.2
2.9 • 0.2
3.4 • 0.1
3.0 • 0.1"
2.2 • 0.1
1.9 • 0.1
Phosphatidic acid 2.5 mo 26.5 mo 0.7 0.5 13.4 2.6 33.3 11.4 0.7 0.2
1.0 0.6 19.2 2.9 32.4 11.7 0.5 0.2
0.3 10.8 0.4 1.7 0.5 0.7 22.2 0.6 0.6
0.3 18.5 0.3 0.8 0.5 0.2 10.3 0.4 0.3
2.1
1.6
Lipids were preparatively isolated by means of thin layer chromatography and their fatty acid composition was analyzed by gas liquid chromatography of fatty acid methyl esters. Results are presented as mean values + S.D. from 3 samples, each containing 6-8 retinas. Average unsaturation represents the average number of double bonds in fatty acids per mole of lipid. *, Signiaficant effects of aging (P < 0.05 or less}. Phosphatidic acid was analyzed after combining the three samples of each group.
TABLE 3 Tetra-, Penta- and Hexaenoic Fatty Acids of Rat Retina Phosphatidylcholine Fatty acid Up to 20:4n-6 20:4n-6 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3 a 24:5n-3 24:6n-3 b 26:5n-3 26:6n-3 c 28:5n-3 28:6n-3 30:4n-6 30:5n-3 30:6no3 32:4n-6 32:5n-3 32:6n-3 34:4n-6 34:5n-3 34:6n-3 36:4n-6 36:5 + 36:6n-3 Total VLCPUFA Mol% Wt%
Controls (mol% • 10)
Aged (mol% X 10)
750.75 54.00 0.20 4.10 3.50 1.80 170.10 1.20 2.70 0.06 0.40 0.06 0.21 0.02 0.06 0.06 0.08 1.10 5.00 0.20 0.70 2.90 0.01 0.08
804.40 56.70 1.40 4.90 1.30 2.50 118.90 1.10 1.40 0.15 0.30 0.06 0.15 0.01 0.06 0.02 0.10 0.80 3.20 0.20 0.60 1.70 0.01 0.06
1.56 2.41
0.99 1.53
VLCPUFA, very long chain polyunsaturated fatty acid. Methyl esters from the phosphatidylcholine samples whose composition is shown in Table 2 were combined and injected in a concentrated form to facilitate detection and quantitation of VLCPUFA. Total VLCPUFA is the sum of C24 to C3~ polyenes. a,b, cCoelute with minor amounts of 24:4n-6, 26:4n-6 and 28:4n-6, respectively.
to show the series of very long chain polyenoic fatty acids t h a t e l u t e a f t e r 22:6 {2). C2,-C36 p o l y e n o i c f a t t y a c i d s m a d e up 2.4% of the fatty acid weight of PC from rat retina. This may seem a minor proportion, but such very long chain polyenes are specifically esterified to some of the P C s {the d i p o l y u n s a t u r a t e d m o l e c u l a r s p e c i e s o f t h e p h o s pholipid) present in photoreceptor membranes (which in t u r n a r e a s m a l l f r a c t i o n o f t h e t o t a l m e m b r a n e s o f ent i r e r e t i n a ) (2). H e n c e , t h e p r o p o r t i o n o f v e r y l o n g c h a i n p o l y e n e s in t h e e n t i r e r e t i n a P C is s m a l l b e c a u s e t h e y are " d i l u t e d " w i t h t h e P C s f r o m m a n y cell m e m b r a n e s t h a t d o n o t c o n t a i n s u c h f a t t y a c i d s . E v e n so, s o m e v e r y l o n g c h a i n p o l y e n e s , l i k e 32:6 a n d 34:6, o c c u r i n h i g h e r p r o p o r t i o n s t h a n s o m e o f t h e p o l y e n e s l i s t e d b e t w e e n 20:4n-6 a n d 2 2 : 6 n - 3 s h o w n i n T a b l e 3. The sum of very long chain polyenoic fatty acids of PC decreased in aged retinas, but some of them contributed m o r e t h a n o t h e r s t o t h i s e f f e c t ( T a b l e 3). T h u s , c o n s i s t ent with the effect observed for the "shortest" hexaene (22:6n-3), all l o n g e r n-3 h e x a e n e s w e r e a l s o d e c r e a s e d . V e r y l o n g c h a i n (n-3) p e n t a e n e s a n d {n-6) t e t r a e n e s w e r e less affected, as was also the case for the shortest polye n e s o f t h e r e s p e c t i v e s e r i e s , s u c h a s 20:5n-3 a n d 20:4n-6. This fits into the general tendency observed for the p o l y e n o i c f a t t y a c i d s o f all r e t i n a l g l y c e r o p h o s p h o l i p i d s , w h i c h a r e s u m m a r i z e d i n T a b l e 4: a) t h e r a t i o b e t w e e n n-3 a n d n-6 p o l y e n e s d e c r e a s e d w i t h a g e , n o t d u e t o a n inc r e a s e i n n-6, b u t t o a d e c r e a s e i n n-3 p o l y e n e s ; b) a m o n g n-3 p o l y e n e s , t h e n-3 h e x a e n e / p e n t a e n e r a t i o d e c r e a s e d w i t h a g e , d u e p r i m a r i l y t o a d e c r e a s e i n n-3 h e x a e n e s ; c) t h e a b s o l u t e a m o u n t s o f t o t a l p o l y e n e s a s w e l l a s t h e i r percentages in lipids decreased with age, showing that no polyene was synthesized to substitute for the depleted 22:6; d) t h e e f f e c t o n p o l y e n e s m a i n l y a c c o u n t e d f o r t h e
LIPIDS, Vol, 22. No. 4 (1987)
256 N.P. ROTSTEIN ET AL. TABLE 4 Summary of the Effects of Aging on Polyenoic Fatty Acids of Retina Glycerophospholipids
PC (mol%)
Total polyenes n-6 Polyenes n-3 Polyenes n-3/n-6 Polyenes n-3 Pentaenes n-3 Hexaenes n-3 Hexa/pentaenes Total polyenes (nmol/mg protein) a 22:6n-3 (nmol/mg protein) a
EGP (tool%)
PS (mol%)
PI (mol%)
PA (mol%)
2.5 mo
26.5 mo
2.5 mo
26.5 mo
2.5 mo
26.5 mo
2.5 mo
26.5 mo
2.5 mo
26.5 mo
26.0 7.3 18.7 2.6 0.5 18.2 36.4
20.8* 7.5 13.3" 1.8" 0.7 12.6" 18.0"
55.8 14.1 41.7 3.0 0.9 40.8 45.3
52.9 14.9 38.0 2.6 1.6" 36.4 22.8*
59.6 9.4 50.2 5.3 2.7 47.5 17.6
51.9" 8.2* 43.7* 5.3 2.8 40.9* 14.6"
51.2 47.6 3.6 0.1 0.5 3.1 6.2
45.0* 41.2" 3.8 0.1 0.9 2.9 3.2
38.5 14.0 24.5 1.8 1.7 22.8 13.4
32.1 20.6 11.5 0.6 0.9 10.6 11.8
20.7
13.7"
32.1
23.6
12.2
8.5*
4.3
3.6
1.1
0.8
13.5
7.8*
23.2
16.0"
9.2
6.4*
0.3
0.2
0.6
0.3
PC, phosphatidylcholine; EGP, ethanolamine glycerophospholipids; PS, phosphatidylserine; PI, phosphatidylinositol; PA, phosphatidic acid. Total polyenes is the sum (mol%) of all polyenes detected in fatty acid composition analyses. The fatty acids were grouped as indicated to calculate the ratios. *, Significant effects of aging (P < 0.05 or less). aAmounts estimated as (nmol fatty acid(s)/nmol lipid) X (nmol lipid/mg protein). tendency shown b y the m a j o r phospholipids to decrease their concentrations in aged retinas (Table 1), since other f a t t y acids were affected to lower extents. The incorporation of [1-'4C]docosahexaenoate in retina lipids was then studied to determine whether the age-dependent defect in 22:6 was due to an impairment of the enzymes involved in the t u r n o v e r of their 22:6-containing species and was c o m p a r e d to t h a t of [2-3H]glycerol in search for possible effects of aging on the de novo synthesis of such lipids.
General features of the incorporation of [3H]glycerol and ['4C]docosahexaenoate in retina lipids. After incubating retinas with these precursors, the incorporation of [~4C]22:6 was faster and more efficient t h a n t h a t of [3H]glycerol (Table 5). One of the i m p o r t a n t factors determining this result was t h a t the f a t t y acid was likely to be present in m u c h larger concentrations in the tissue t h a n glycerol, simply because it is relatively more "soluble" in the retinal lipid m e m b r a n e s t h a n in the medium, as opposed to the water-soluble precursor. In the present experiments, the concentration of free [~C]22:6 in the incubation medium was indeed higher t h a n t h a t of glycerol (5 vs 0.16 nmol, respectively, were offered to the tissue). To account for this difference in the concentration of precursors, the a m o u n t of incorporated glycerol was in all cases multiplied b y a factor of 31.25, b u t even after this correction the 14C/3H ratios were always greater t h a n 1 (Tables 5-7). Such correction, of course, cannot level off the possible differences in intracellular concentrations attained (which are ignored) nor the possible differences in compartmentation. The incorporation of [~H]glycerol into r a t retina lipids followed the general routes t h r o u g h which the de novo synthesis of glycerol-containing lipids occurs in m o s t v e r t e b r a t e tissues. Thus, at early incubation times (10 min), p h o s p h a t i d a t e was the lipid concentrating m o s t of the label, followed b y diacylglycerols and phosphatidylinositol (PI) (Table 6). While P I rapidly attained a plateau, the synthesis of PC continued t h r o u g h o u t the incubation interval, this lipid being the predominant product at long LIPID& Vol. 22, No. 4 (1987)
~~
'~ o]
o
PA
'
Pl
PS
EGP
PCf,,/~
DG
T6
Pl
PS
EGP
~
DO
TG
'
2o ~'o
zo ~o
zo ~o 2o ~o 20 4o incubation time (rain)
2o ~o
2o
4o
FIG. 1. Comparison of the time-course of the distribution of [3H]glycerol and [14C]docosahexaenoate among lipids of control (circles) and aged (triangles) retinas. Results are percentages of the total incorporated activity in each case, calculated from the mean values in Tables 5 and 6. PA, phosphatidic acid; PI, phosphatidylinositol; PS, phosphatidylserine; EGP, ethanolamine glycerophospholipids; DG, diacylglycerols; TG, triacylglycerois.
incubation times. M o s t of the difference in total [3H]glycerol incorporated between 20- and 50-min incubations (Table 5) was accounted for by the labeling of PC {Table 6). The relationships between the various lipids are readily apparent when represented on a relative basis (Fig. 1). The contribution of p h o s p h a t i d a t e and diacylglycerols to the total label in lipids decreased with time, in favor of the final products. A large proportion of the t o t a l free ['4C]22:6 added to the media was recovered in the {comparatively very small volume of) tissue after incubations (Table 5). The esterification of free 22:6 b y the retina was highly efficient, as indicated b y the fact t h a t a large proportion of the total label in the tissue was recovered in lipids (Table 5). In cont r a s t to the redistributions observed for the [3HI label a m o n g lipids (Fig. 1), [14C]22:6 was p r e d o m i n a n t l y incorp o r a t e d in PC at all incubation times analyzed (40-50% of the esterified label). This indicates t h a t m o s t of the
257 A G I N G AND R E T I N A D O C O S A H E X A E N O A T E
TABLE 5
Effect of Aging on the In Vitro Incorporation of [1-14ClDocosahexaenoate and [2-3H]Glycerol in Rat Retinal Lipids [1-'~C]Docosahexaenoate
Control rats
Aged rats
Incubation time (rain)
Free
Esterified
Total ['4C]22:6a
Esterified/free
[2-~H]Glycerol
10 20 50
952 • 125 914 • 135 769 • 202
45 • 20 205 • 15 229 • 20
1038 • 70
0.05 0.22 0.29
9 • 1 78 • 8 106 • 8
5.0 2.6 2.2
10 20 50
972 • 100 746 • 120 7 0 7 • 174
117 • 11 352 • 13 369 • 26
1087 • 12
0.12 0.47 0.52
20 • 7 79 • 17 110 • 14
5.9 3.3 3.3
"CPHb
The incorporations are expressed as pmol ['4C]22:6 or [3H]glycerol in lipids/mg of retinal protein (mean values • S.D. from 3 samples, each containing the two retinas of a rat). Control and aged retinas were 2.5 and 26.5 m o n t h s old, respectively. The retinas were incubated for the specified intervals with b o t h precursors, added simultaneously. The amount of incorporated [2-~H]glycerol was multiplied by a factor of 31.25 to account for the difference in the amounts of I14C]22:6 and ~3H]glycerol added to the incubation media (5 and 0.16 nmol, respectively). aFree + esterified. bEsterified [1-14C]docosahexaenoate/[2-3H ]glycerol.
TABLE 6
Labeling of Retina Lipids by [2-3H]Glycerol and [1-'4C]Docosahexaenoate [1-'4C]Docosahexaenoate
[2-3H]Glycerol 10 min
20 rain
50 min
10 min
1.1 -+ 0.1 0.7 • 0.1 0.5 • 0.5
19.2 ___ 1.6 9.4 • 0.5 7.1 • 3.3
43.2 __ 3.7 17.2 _+ 3.7 10.2 • 1.1
17.3 • 0.9 8.4 __ 1.0 2.9 __ 0.9
Phosphatidylinositol Phosphatidic acid
1.3 ___ 0.5 2.7 • 0.2
7.8 _+ 2.8 17.0 • 2.9
9.1 _ 0.4 15.3 __ 2.0
3.1 _+ 1.7 7.5 + 1.0
12.5 • 27.7 --
2.3 8.8
14.6 ___ 4.8 26.8 • 4.1
Diacylglycerols Triacylglycerols
2.2 • 0.6 1.0 • 1.0
14.9 ___ 0.1 3.2 • 1.2
8.5 __ 0.6 2.8 _+ 2.6
4.2 • 3.1 1.9 -- 0.6
12.3 ___ 0.6 5.1 - 2.3
7.1 __- 0.4 2.7 • 2.3
P C / E G P ratio
1.5 • 0.1
2.0 __ 0.5
2.5 __ 0.8
2.1 • 0.3
3.6 -- 0.2 0.9 --- 0.2 2.8 • 0.1
24.1 • 0.7 7.1 • 0.1 10.0 __ 2.0
41.5 • 0.4 12.0 • 0.8 14.5 • 2.3
47.7 • 4.5 12.0 + 4.0 10.4 __ 0.9
Phosphatidylinositol Phosphatidic acid
2.7 • 0.8 5.6 • 0.4
8.3 __ 2.5 11.6 • 2.9
8.8 • 2.1 13.4 • 2.3
10.6 -- 3.4 15.1 __ 1.0
18.7 • 38.8 •
2.5 6.0
14.0 __- 4.4 35.0 +_ 2.2
Diacylglycerols Triacylglycerols
2.9 + 1.2 1.5 --- 1.1
10.9 • 0.4 4.8 • 4.7
13.0 • 3.6 7.1 __ 3.6
12.2 • 4.8 6.8 + 6.9
20.1 + 6.0 20.1 • 21.0
22.8 + 8.8 21.7 __- 13.6
P C / E G P ratio
3.9 +- 0.5
3.5 +_ 0.5
4.0 -'!- 1.8
Control rats Phosphatidylcholine Ethanolamine glycerophospholipids Phosphatidylserine
Aged rats Phosphatidylcholine Ethanolamine glycerophospholipids Phosphatidylserine
3.4 _+ 0.03
20 min
50 rain
92.3 -- 10.9 41.2 • 3.0 13.7 _+ 5.7
2.3 --
0.3
164.5 • 20.4 52.8 • 7.8 37.7 • 7.4
3.2 •
0.8
109.2 __- 1.3 43.2 __- 3.2 25.5 _+ 3.0
2.5 __- 0.2 182.4 • 52.8 • 40.2 •
4.4 4.3 9.2
3.1 +_ 0.2
Lipids labeled in the retina with b o t h radioactive precursors were separated by thin layer chromatography. The figures represent the incorporation into each lipid class {pmol/mg retinal protein}. Details as in Table 5.
available 22:6 was incorporated directly into preexisting PC molecules through acyl exchange reactions, rather than through the slower neobiosynthetic reactions. Howe v e r , l a b e l f r o m t h e f a t t y a c i d w a s i n a l l g l y c e r o l i p i d s , inc l u d i n g p h o s p h a t i d a t e {PA) a n d d i a c y l g l y c e r o l , f o r p a r t of the label in major phospholipids, particularly at long i n c u b a t i o n t i m e s , m a y b e c o n t r i b u t e d b y {the i n c o r p o r a t i o n of} t h e s e l a b e l e d i n t e r m e d i a t e s . T h e s p e c i f i c r a d i o -
activity of PA was the highest among phospholipids with e i t h e r g l y c e r o l o r 2 2 : 6 { T a b l e 7), w h i c h s u p p o r t s t h i s possibility. The differences in rates of incorporation of both precurs o r s b e c o m e e v e n l a r g e r i n f a v o r o f 2 2 : 6 if o n e t a k e s i n t o account that while [~H]glycerol is introduced in the s k e l e t o n o f all k i n d s o f l i p i d m o l e c u l a r s p e c i e s f r o m d i s a t u r a t e d t o d i p o l y u n s a t u r a t e d / 9 } , [14C]22:6 o n l y l a b e l s
LIPtDS, Vol. 22, No. 4 (t987)
258
N.P. ROTSTEIN ET AL. TABLE 7 Aging Effects on Average Specific Radioactivities of Retina Glycerophospholipids
[2-3H]Glycerol Incubation time(rain) PCa EGP PS PI PA
[1-1'C]Docosahexaenoate PC
EGP
PS
PI
[1-14C]Docosahexaenoate PA
PC
Aged rats Agedrats/ con~olrats
10 20 50 10 20 50 10 20 50
7 121 272 27 185 315 3.9 1.5 1.2
6 82 149 10 80 135 1.7 1.0 0.9
12 174 250 85 305 442 7.0 1.8 1.8
78 470 545 167 512 543 2.1 1.1 1.0
474 2980 2680 1077 2230 2580 2.3 0.7 1.0
109 608 720 362 1250 1500 3.3 2.1 2.1
73 360 374 134 593 660 1.8 1.6 1.8
171 335 625 317 1150 1225 4.5 3.4 2.0
PS
PIX10 -~ PA•
-3
(fraol [14C]22:6/nmol 22:6)c
(fmol ~H or l~C/nmol lipid p)b Contr~ rats
EGP
185 750 870 650 1150 864 3.5 1.5 1.0
1316 4860 4700 2904 7460 6730 2.2 1.5 1.4
320 1790 2120 1520 5250 6300 4.8 2.9 3.0
90 450 465 185 825 915 2.1 1.8 2.0
80 370 690 405 1465 1560 5.0 2.3 2.3
3.0 12.1 14.0 11.2 19.9 14.9 3.7 1.6 1.1
3.0 11.0 10.6 14.1 36.2 32.7 4.7 3.3 3.1
apc, phosphatidylcholine; EGP, ethanolamine glycerophospholipids; PS, phosphatidylserine; PI, phosphatidylinositol; PA, phosphatidic acid. bThe specific radioactivity of each lipid class was calculated as (fmol 3H or 14C in lipid/mg protein)/(amount of lipid/mg protein). cSpecific radioactivities of 22:6-containing species within each lipid class, calculated as (fmol ['4C]22:6/nmollipid [fmol 3H or 14Cin lipid/rag protein])/(nmol of 22:6/nmol lipid).
the 22:6-containing species of each retinal lipid (namely hexaenoic and dipolyunsaturated species [10]). The highest specific radioactivities, whether expressed per mole of lipid or per mole of 22:6 in lipid {Table 7), were attained b y P A and P I with b o t h precursors. A m o n g the three major m e m b r a n e phospholipids (PC, E G P and PS), the highest turnover rates of 22:6 (i.e., the exchange of labeled for unlabeled f a t t y acid} were attained b y docosahexaenoat~containing species of PC. This is noteworthy, since b o t h PS and E G P are richer in 22:6 t h a n PC {Table 2).
group {Table 7). The selective stimulation of the turnover of 22:6 in PC and PS became even more a p p a r e n t when the specific radioactivities were expressed as fmol [14C]22:6 incorporated per mole of 22:6 in each lipid {Table 7), since the latter tended to decrease in all lipids of aged retinas (Table 2) b u t especially in these two phospholipid classes. DISCUSSION
Effects of aging on retinal lipid labeling with [3HI- A m a r k e d decrease in the levels of 22:6n-3 along with glycerol and [~'C]22:6. In the presence of equal a m o u n t s other polyenoic f a t t y acids occurs in the glycerophosphoof free [~4C]22:6, retinas from aged r a t s incorporated a b o u t twice as m u c h f a t t y acid in their lipids t h a n those from y o u n g animals (Table 5). The labeling of all lipids b y the f a t t y acid was stimulated {Tables 6 and 7), b u t PC and PS showed the largest increases in relation to other lipids (Fig. 1). The incorporation of [3H]glycerol was m u c h less affected b y aging t h a n t h a t of [~'C]22:6 (Table 5). No significant differences were observed in the total glycerol incorporated after 20- and 50-min incubation, even t h o u g h a stimulation was a p p a r e n t at short incubation times (10 min). This m a y have been p r o v o k e d b y the presence of unesterified [1"C]22:6 in the tissue since, coincidentally, the lipids whose labeling with [3H]glycerol was m o s t s t i m u l a t e d {especially early on) were also PC and PS (Table 6, Fig. 1). Of the two zwitterionic phospholipids, synthesis of PC was relatively more stimulated t h a n t h a t of E G P (see the P C / E G P ratios in Table 6). Similarly, of the two acidic phospholipids, labeling of PS increased more t h a n t h a t of P I (note t h a t the labeled P S / P I ratios were 0.4, 0.9 and 1.1 for controls and 1.0, 1.2 and 1.6 for aged retinas after 10-, 20- and 50-rain incubations, respectively, with glycerol}. Such changes were not due to a depressed synthesis of E G P or P I (Tables 6 and 7) b u t to a relatively faster formation of PC and PS. The specific radioactivities of [l~C]22:6-1abeled lipids were in all cases larger in the retinas from the aged r a t LIPIDS, Vol. 22, No. 4 (1987)
lipids of retina as a consequence of aging {Table 4). The phospholipids containing the largest percentages of docosahexaenoate (PC, E G P and PS) are those t h a t exhibit the largest q u a n t i t a t i v e decreases of this f a t t y acid {Tables 2-4}. This is a p p a r e n t l y not c o m p e n s a t e d b y increases in other f a t t y acids and is manifested in the tendency these phospholipids show to decrease their concentrations when t h e y are expressed as nmol lipid/mg of retinal protein (Table 1). Phospholipids like P I and D P G , which contain m u c h lower levels of 22:6 and other n-3 polyenoic f a t t y acids, are virtually unchanged, as is also the case with Sph (Table 1), which does not contain any polyenes. I t is a p p a r e n t t h a t aging decreases the ability of retina to make 22:6-containing species of phospholipids. When retinas are incubated with [1-14C]docosahexaenoate, however, it is observed t h a t the esterification of the fatt y acid into lipids is m a r k e d l y stimulated with aging {Table 5). The lipids whose labeling is m o s t stimulated are PC and PS {Tables 6 and 7), precisely the classes undergoing the largest decreases in 22:6n-3, as well as in longer n-3 hexaenoic f a t t y acids {Tables 2 and 3). The incorporation of [3H]glycerol and [14C]22:6 into lipids shows t h a t the de novo biosynthetic machinery as well as the enzymatic m e c h a n i s m s involved in the turnover of 22:6 in preexisting phospholipid molecules are not responsible for the observed decreases in docosahexaenoate. On the contrary, aged retinas seem to work as
259
AGING AND RETINA DOCOSAHEXAENOATE though they were more "avid" for 22:6 than their younger counterparts (Tables 5-7), i.e., as though they had developed a higher affinity for the fatty acid. The results allow us to conclude that the levels of 22:6-containing species of lipids are decreased during aging simply because there is less 22:6 available in the retina, since when the fatty acid is provided such enzymes work to attain, and even surpass, their fullest capacity. That the decrease of 22:6 in lipids is not due to phospholipase As activation during aging is supported by the fact that the levels of free fatty acids did not differ between young and aged retinas. The total free fatty acid pool size in the retinas whose composition is shown in Tables 1 and 2 was 16.2 _+ 3.1 and 17.9 ___ 3.7 nmol/mg of protein, respectively, of which only 1.8 __+ 0.2% and 2.3 +_ 1.1% was 22:6n-3, and 4.3 ___1.4% and 1.7 _+ 0.4% was 20:4n-6, respectively (mol% of the total unesterified fatty acids). Even when these are not strictly "endogenous" levels and percentages of free fatty acids, since these are known to be quickly released from excitable tissue lipids due to postdecapitation ischemia (ll), no significant effect of age was observed, i.e., no accumulation of polyunsaturated fatty acids occurs in aged retinas, which may account for the decreases observed in phospholipids. Therefore, neither the synthetic nor the degradative processes of phospholipids themselves are responsible for the decreased levels of polyenoic fatty acids displayed in their composition. There are at least two possibilities to explain the decreased levels of polyenoic fatty acids in lipids of aged retinas: either they are destroyed at higher rates, or they are synthesized less efficiently. Concerning the first alternative, reactions leading to the formation of several peroxidation products and free radicals from polyunsaturated fatty acids may be involved. In fact, these deleterious products do accumulate in senescent tissues, and a debilitation of the defense mechanisms of cells to dispose of them has been proposed to be an important factor contributing to the process of aging (12-14). The susceptibility of lipids to peroxidation runs parallel to their content in polyenoic fatty acids, which makes brain, and especially retina, lipids particularly prone to such damaging reactions. Docosahexaenoate is highly labile, since the larger the number of double bonds, the larger the number of possible hydroperoxides that may be produced, and the greater the rate constants of the reactions leading to free radical formation (15). Products of lipid peroxidation in turn may react with many cell molecules (13,16), giving rise to lipid-lipid, lipid-protein and proteinprotein crosslinking. Such crosslinked products may contribute to the decrease in fluidity observed in many cell membranes during aging (17,18}. Interestingly, the micr(~ viscosity of synaptosomal cortex membranes increases with age, the increase being higher for the hydrophobic than the hydrophilic region of the membrane, which supports this possibility (19}. It is obvious at present that many chemical processes of completely differing natures may contribute during aging, with effects that are all translated as a change in this bulk physical property of membranes, including increases in cholesterol content, decreases in unsaturated fatty acids, alterations in phospholipid headgroup ratios, formation of crosslinked products and others. Concerning the second possibility, i.e., that decreased
levels of polyenoic fatty acids in retinal lipids may result from a decreased rate of synthesis of these highly unsaturated acyl moieties, two possibilities may be considered: first, that the enzymes that synthesize them (from essential fatty acids of the n-3 and n-6 series) work normally, but lower amounts of such precursors are available, and second, that such precursors are available, but some of the enzymatic steps involved in their further conversions are impaired. It is well established that the synthesis of 22:6n-3 follows the route 18:3n-3 18:4n-3 ~ 20:4n-3s 20:5n-3 ~ 22:5n-3~ 22:6n-3. Similarly, the synthesis of 22:5n-6 follows the route 18:2n-6 18:3n-6 ~ 20:3n-6 ~ 20:4n-6 ~ 22:4n-6 ~ 22:5n-6. Steps 1, 2 and 3 are catalyzed by A6, As and A4desaturase systems, respectively, the other reactions being elongations. Much more is known on the regulation of & and A5than of s desaturases (20}, but there is strong evidence that the desaturations rather than the elongations are rate-limiting steps in the sequences (21,22}. [1-'4C]20:5n-3 is readily transformed into ['4C]22:5n-3 and this into ['"C]22:6n-3 in the retina in vivo a few minutes after the injection of the precursor into the eye (23). This indicates that the retina does not rely on other organs (e.g., the liver} for the supply of 22:6, but is able to synthesize its own 22:6 from other (n-3} fatty acids (which, of course, must be available}. The levels of n-3 pentaenes in retina lipids were much less affected by aging than n-3 hexaenes (Table 4), indicating that there was no defect in the availability of essential n-3 fatty acid precursors like 18:3n-3. Moreover, there is an important difference between the effect of aging shown here and that of essential fatty acid deficiency (24}. Thus, during 18:2n-6 deficiency, 20:4n-6 is decreased in tissue lipids, but replaced by 20:3n-9 (made from oleate). During 18:3n-3 deficiency, 22:6n-3 decreases in lipids, being replaced by 22:5n-6 (made from linoleate). None of these compensatory mechanisms was observed in the aged retina. Moreover, the retina is known for the tenacity with which it holds 22:6 in its lipids, since generations of n-3 fatty acid precursor-deficient diets must elapse before a significant decrease of 22:6 can be observed in retina, as opposed to the rapid "adaptation" of liver, kidney and brain (25, 26). When the deficiency is eventually achieved, however (after at least two generations}, the decreased 22:6n-3 is quantitatively replaced by 22:5n-6 (26-28}. This is not the case with the aged retina, where both 22:6n-3 and 22:5n-6 decrease (Tables 2 and 3). It is quite coincidental that the two fatty acids whose synthesis is catalyzed by A, desaturase are the ones that undergo the most significant and consistent decreases, as illustrated in Table 8 by the ratios between the amounts (in nmol/mg protein} present in aged vs control retinas, as calculated from the data in Tables 1 and 2. This strongly suggests that, rather than a decreased availability of 18:3n-3 (and 18:2n-6) in the retina, an impairment of the/~ desaturase system is probably responsible for the decreased levels of 22:6n-3 (and 22:5n-6) observed in retina lipids as a consequence of aging. It is noteworthy that, in addition to 22:6n-3, other n-3 hexaenoic fatty acids are also decreased in aged retinas. This is clearly evident for PS (24:6n-3, Table 2) and for PC (Table 3), which contains a whole series of polyenoic fatty acids ranging from 24 to 36 carbons. The very long chain polyenes of PC fit into the general tendency observed LIPIDS, Vol. 22, No. 4 (1987)
260
N.P. ROTSTEIN ET AL. TABLE 8 Ratios Between Amounts {in nmol/mg Protein} of Fatty Acids in Aged vs Control Retinas (Calculated from Data in Tables I and 2)
20:4n-6 22:4n-6 22:5n-6 20:5n-3 22:5n-3 22:6n-3
PC
EGP
PS
PI
0.9 1.0 0.4 4.1 1.2 0.6
0.9 0.7 0.3 1.5 1.1 0.7
1.0 0.6 0.2 0.8 0.9 0.7
0.8 1.0 <0.1 1.5 >1.6 0.9
PC, phosphatidylcholine; EGP, ethanolamine glycerophospholipids; PS, phosphatidylserine; PI, phosphatidylinositol.
for other polyenes, namely, t h a t n-3 hexaenes (as 22:6n-3) decrease, and t h a t t h e y do so to a larger extent t h a n n-3 pentaenes (as 20:5 and 22:5n-3) and n-6 tetraenes {as 20:4 or 22:4n-6). Very long chain tetraenes of vertebrate retina belong to the n-6 series and hexaenes to the n-3 series, and there are v e r y long chain pentaenes from b o t h the n-3 and n-6 families (2,3}. The latter are in negligible a m o u n t s in r a t retina b u t occur in larger proportions in r a b b i t and chicken retina PC (2). I n chickens, it was observed t h a t v e r y long chain (n-6) pentaenes increase while v e r y long chain In-3) hexaenes decrease under dietary conditions t h a t lead to an increased 22:5n-6/ 22:6n-3 ratio (2). The c o n c o m i t a n t decrease of v e r y long chain hexaenes and 22:6 observed here in r a t retina PC is consistent with the idea t h a t less 22:6 is produced during aging, since the i m p a c t is felt in the " s h o r t e s t " hexaene as well as in the s u b s e q u e n t l y longer hexaenes t h a t are synthesized b y successive elongations of 22:6 (Rotstein, N.P., and Aveldafio, M.I., unpublished work). This fits into what is known on the synthesis of polyenoic f a t t y acids, i.e., t h a t the rate-limiting steps are those catalyzed b y desaturases, r a t h e r t h a n those involving elongations, which mainly depend on the availability of the respective precursors (21), in this case 22:6. If the decrease in polyenes were exclusively due to increased r a t e s of peroxidation during aging, one would p r o b a b l y not observe such trends of selectivity toward hexaenes and, especially, there would be no a p p a r e n t reason for a decrease in 22:5n-6 much larger than t h a t of 22:5n-3, since b o t h f a t t y acids h a v e identical n u m b e r of carbons and double bonds. I t is evident from the ["C]22:6 incorporation exp e r i m e n t s t h a t if docosahexaenoate is available, it is efficiently t a k e n up b y the aged retina, and it is m o s t actively introduced into the phospholipid classes t h a t show the largest decreases, not only of 22:6n-3 b u t of longer n-3 hexaenes. These o b s e r v a t i o n s are interesting f r o m a functional point of view, since there is evidence t h a t n-3 p o l y u n s a t u r a t e d f a t t y acids are necessary for the normal electrical response in visual excitation. Thus, a decrease of retina 22:6n-3 attained b y dietary manipulation results in a change of the c o m p o n e n t of the e l e c t r o r e t i n o g r a m t h a t is generated b y photoreceptors (27). Docosahexaenoate (27) as well as v e r y long chain polyenoic f a t t y
LIPIDS, VoL 22, No. 4 (1987)
acids (2) are highly c o n c e n t r a t e d in the lipids of photoreceptor membranes. Therefore, a decrease in these f a t t y acids, which is observed even when the lipids of the entire retina are analyzed, m u s t play an i m p o r t a n t role a m o n g the causes of vision impairment, which almost inv a r i a b l y accompanies senescence. The avidness with which aged retinas incorporate [14C]22:6 in their 22:6depleted lipids s u g g e s t s the exciting possibility t h a t an adequate supply of this f a t t y acid in the diet {rather t h a n of its precursors) m i g h t help prevent, or at least delay, one of the m a n y biochemical alterations t h a t set in with aging. REFERENCES
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