789

Age-Dependent Accumulation of Phosphatidylcholine Hydroperoxide in the Brain and Liver of the Rat Teruo Miyazawa*, Toshihide Suzuki and Kenshiro Fujimoto Department of Applied Biological Chemistry, Tohoku University, Tsutsumidori, Sendai 981, Japan

Age-related changes in phosphatidylcholine hydroperoxide (PCOOH) content as an index for oxidative membrane lipid damage were determined by high-performance liquid chromatography using chemiluminescence detection. Brain and liver PCOOH content increased significantly in male and female rats with age. The brain PCOOH content of male 18-month-old rats was 4.4 times that of 1-month~fld rats, and that of female 18-month-old rats was 3.5 times that of 1-month-old females. The fiver PCOOH content of the male 1&month-old rats was 9.3 times that of the 1month-old; and of the female l&month~)ld rats was 4.7 times that of the 1-month-old. PCOOH levels in heart and lung did not show age dependency. In both brain and fiver (but not in heart and lung), the phosphatidylcholine content significantly decreased upon aging. The results indicate that oxidative deterioration, such as phosphofipid hydroperoxidation, is prevalent in the membrane lipids of brain and fiver of the rat due to aging. Lipids 28, 789-793 (1993).

Peroxidation of membrane lipids has been implicated as one of the basic mechanisms of ag~related pathological changes (1-3). The thiobarbituric acid (TBA) assay has previously been employed to estimate lipid peroxides in human plasma (4) and in tissues of rodents as a function of age (5-11). Several investigators have reported an increase in TBA reactants in rat brain (5,8,9,11) and liver (8,9,11) with aging whereas others could not show such changes (6,7,10). These inconsistencies may well be attributable to methodological problems, because the TBA assay also responds to alde~ hydes, amino acids and carbohydrates unrelated to lipid peroxides (12,13). Alternatively, expiration of hydrocarbon gas by aged rats (14), conjugated diene absorption of tissue lipids upon aging (15) and ag~related fluorescence pigment formation (16) have been measured to follow membrane lipid hydroperoxide formation. However, to accurately assess the degree of lipid hydroperoxidation in biological membranes, the direct measurement of primary peroxidation products, such as phospholipid hydroperoxides, is most desirable A new, sensitive and selective assay of phospholipid hydroperoxides by high-performance liquid chromatography (HPLC) combined with chemiluminescence (CL) detection was first described by Miyazawa and colleagues (17-20). This CL-HPLC method was successfully applied to measure phospholipid hydroperoxides present in human plasma (19, 21), serum lipoproteins (22) and in liver and brain of rodents (20,23,24). More recently, this method was used to establish that the phosphatidylcholine hydroperoxide (PCOOH) con-

*To whom correspondence should be addressed. Abbreviations: CL, chemiluminescence;DHA, docosahexaenoicacid; HPLC, high-performanceliquid chromatography;PC, phosphatidylcholine;PCOOH,phosphatidylcholinehydroperoxide;PEOOH, phosphatidylethanolaminehydroperoxide;TBA, thiobarbituricacid; TL, total lipids; TLC, thin-layerchromatography. Copyright 9 1993 by the American Oil Chemists' Society

tent of Drosophila melanogaster flies is reciprocally propor ~ tional to the flies' life span (25,26). PCOOH accumulation in cultured human diploid cell~ upon reaching cellular population doubling levels, was also measured by this method (27). A similar HPLC method with CL detection was independently described by Frei et aL (28), although these authors could not detect phospholipid hydroperoxides in human blood plasma due to methodological limitations. In the present paper, we primarily determined the agerelated changes that occur in PCOOH levels in the internal organs of the rat using the CL-HPLC method to follow the oxidative damage of membrane lipids in this in vivo rodent

system.

MATERIALS AND METHODS Animals. Male and female Sprague-Dawley rats (n = 42) were fed a standard laboratory diet (F-2 pellet rations; Funabashi Farm Co., Chiba, Japan; containing 5 mg of vitamin E per 100 g of diet) ad libitum for 1, 7, 12 and 18 mon for male rats (body weight, 140, 557, 678 and 722 g, respectively; n = 6 for each group); and for 1, 7 and 12 mon for female rats (body weight, 112, 334 and 443 g, respectively; n = 6 for each group). Groups of three rats each were housed in stainless steel cages and maintained at 25~ on a 12 h (8 a.m. to 8 p.m.) light-dark cycle The rats were fasted for 20 h prior to dissection and were sacrificed by exsanguination under light diethyl ether anesthesia. The livers were perfused in situ with ice-cold 0.15 M saline, and then the brain, liver, heart and lung were removed. Total lipid (TL) extraction. TL were extracted with a mixture of chloroform and methanol (2:1, vol]vol) (29) from brain, liver, heart and lung as described previously (23,24). C L - H P L C The CL-HPLC system and conditions for measuring PCOOH in total lipids from rat tissues were essentially the same as those previously described by Miyazawa et aL (23,24). Determination of phosphatidylcholine (PC) and a-tocopheroL The PC content in tissue TL was measured by an Iatroscan thin-layer chromatography (TLC)/flame-ionization detection method using an Iatroscan TH-10 apparatus (Iatron Laboratories Inc., Tokyo, Japan) as described by Hazel (30). o~Tocopherolcontent was measured by the fluorescence-HPLC method (31). Fatty acid analysis. PC was separated from tissue TL lipids by TLC (silica gel 60; Merck, Darmstadt, Germany) using chloroform/methanol]acetic acid/water (25:15:4:2, by vol) as developing solvent (32). Fatty acid methyl esters derived from TL and from PC were prepared by acidcatalyzed transmethylation (33). Methyl esters were extracted with hexane and were analyzed using a Shimadzu GC-8A gas chromatograph equipped with a glass column (300 • 0.2 cm) containing 10% Silar 10C on Chromosorb W (60/80 mesh; Supelc~ Bellefonte, PAL Column temperature was programmed from 170 to 220~ at l~ Fatty acid methyl esters were identified by comparison of their retention times with those of standards. LIPIDS, Vol. 28, no. 9 (1993)

790 T. MIYAZAWA E T AL.

Statistical analysis. The data are expressed as mean values with standard deviations. All data were analyzed using the Student's t-test.

identity was further confirmed by cochromatography with authentic PCOOH. The chemiluminescent peak due to the hydroperoxide group in PCOOH completely disappeared upon reduction of the tissue TL with sodium borohydride to give the corresponding hydroxyl derivative as has been RESULTS previously reported (18-20). As shown in Figure 1, the Figure 1 shows the CL chromatograms of PCOOH from PCOOH peak intensity observed for the brain sample brain TL of young male rats (1-month-old, Fig. 1A) and from 18-month-old rats (767 pmol PCOOH/g brain) was of aged male rats (18-month-old, Fig. 1B). PCOOH (reten- more intense than that for 1-month-old rats (140 pmol tion time at 11.0 min) was clearly separated and detected PCOOH/g brain). By measuring conjugated diene absorpas a single sharp peak by CL detection. This peak had tion (lower chart of Fig. 1), a slight, but insignificant, inthe same retention time as did authentic PCOOH; its crease was observed for 18-month-old rats compared to 1-month-old rats. The lack of significance was probably due to the comparatively low sensitivity of the conjugated diene method. (A) (B) Brain. For both sexes, brain weights, TL and a-tocopherol content at 7, 12 and 18 mon were significantly PCOOH higher than those at one month (Table 1). The brain PC content expressed per brain weight was constant during C C I20 mV .9 o the 18-mon feeding period, but the PC content in the TL O O showed an age-dependent decrease for both sexes. The 9 9 brain PCOOH was increased with aging in both male and female rats. The brain PCOOH content of the 18-monthJ old male rats was 4.4 times that of the 1-month-old, and 0 PCOOH 0 of the 18-month-old female rats was 3.5 times higher than that of the 1-month-old. Liver. Liver weights increased with aging (Table 2). However, TL contents were not age-dependent, except for the 18-month-old female rats that had the highest content. The PC content of TL in males was lower at 12 and 18 mon than at 1 and 7 mon, and in females PC content was lower at 18 mon than at 1 and 7 mon. mTocopherol contents both in male and female rats were not age0.064 AU dependent. Female liver a-tocopherol contents were =o PC O significantly higher than those of male livers for all age groups examined. Liver PCOOH increased proportionally with age in both sexes. The PCOOH content of the 18month-old male rats was 9.3 times higher than that of the 1'2 1'6 () 1-month-old, and that of the 18-month-old female rats was retention time (min) retention time (min) 4.7 times that of the 1-month-old. Female rats had a higher liver PCOOH content at the early ages (1- and 7-monthFIG. 1. C h e m i l u m i n e s c e n c e (CL)-high-performance liquid chromatographychromatogramsof phosphatidylcholinehydroperox- old) compared with male rats, and the 18-month-old ides (PCOOH} from brain total llpids prepared from male rats. Panel females had a lower liver PCOOH content than the A, 1-month-old rats. Panel B, 18-month-old rats. The PCOOH con18-month-old males. centration detected on the chemiluminescent chromatogram corHeart and lung. The heart weights of the 7-, 12- and 18responded to 140 pmol/g brain (A) and 767 pmol/g brain (B). The upmonth old rats were higher than those of the 1-month-old per chart shows CL detection, the lower chart shows conjugated diene rats (Table 3). Lung weights increased with aging. TL (234 nm) detection. UV, ultraviolet.

--y

k___

-

Sg

~

g

L.

,._Z.._

TABLE 1 Changes in Lipid Composition and in a-Tocopherol and Phosphatidylcholine H y d r o p e r o x i d e (PCOOH) Contents of Rat Brain with Aging a Rats (mon) Male 1 7 12 18 Female 1 7 18

Brain weight (g) 1.6 2.1 1.9 1.9

• 44•

0.1 b 0.1 c 0.1 c 0.1 c

1.6 4- 0.1 b 1.8 • 0.1 c 1.8 4- 0.1 c

T o t a l lipids (TL) (mglg brain) 54.2 68.2 71.2 66.7

4- 5,2 b 4- 6.1 c 4- 4.2 c 4-_ 6.8 c

55.3 4- 2.9 b 64.4 4- 4.1 c 71.8 4- 8.5 c

PC (mg/g brain) (mg/g TL) 14.6 14.5 14.6 12.3

4444-

1.6 2.3 1.2 2.2

15.1 4- 1.9 13.2 • 1.0 14.7 4- 2.2

283 219 208 177

__- 28 b + 24 c + 21 c + 22 d

267 ___ 22 b 206 4- 12 c 196 4- 8 c

a-Tocopherol (t~g/g brain) 6.3 12.4 11.4 12.5

4- 0.95 4- 0.5 c 4- 1.7 c 4- 1.9 c

8.7 4- 2.45 14.3 • 0.4 c 15.3 4- 2.1 c

PCOOH (pmol/g brain) 131 168 474 573

-44+-

46 b 495 77 c 144 c

163 4- 415 319 • 56 c 575 4- 73 d

aValue8 are m e a n s __ SD of six r a t s per each age group. M e a n s n o t followed b y the s a m e letter, b,c or d, are significantly different (P < 0.001) w i t h i n each sex.

LIPIDS, Vol. 28, no. 9 (1993)

791 L I P I D H Y D R O P E R O X I D A T I O N IN A G E D RATS TABLE 2

Changes in Lipid Composition and in a-Tocopherol and PCOOH Contents of Rat Liver upon Aging a Rats (mon) Male 1 7 12 18 Female 1 7 18

Liver weight (g) 7.2 21.9 24.1 28.7

• 0.8 c +_ 3.9 d • 2.8 d'e • 3.1 e

5.6 • 0.5 b'c 14.0 _+ 1.9 b,d 14.1 • 2.2 b'd

Total lipids (TL) (mg/g brain) 34.4 30.0 38.0 33.0

• --• •

2.1 3.8 r 4.7 d 7.8

31.2 • 4.0 c 28.7 • 5.2 c 49.6 • 7.0 b'd

PC (mg/g liver) (mg/g TL)

a-Tocopherol (~g/g liver)

15.1 13.3 12.3 9.9

11.2 15.2 9.3 13.2

• +• •

1.7 c 2.5 1.4 1.1 d

13.4 • 2.8 13.6 • 1.2 13.6 • 1.65

468 433 338 309

• -+ • •

10 c 52 c 19 d 64 d

425 • 32 c 449 • 49 c 274 • 41 d

• • • •

PCOOH (pmol/g liver)

3.1 2.5 c 2.3 d 0.9 c

274 674 1983 2558

18.8 • 3.95'c 33.3 • 4.3 b'd 25.1 • 3.6 b'c

• + • •

74 c 19 d 388 e 348 e

409 • 26 b'c 1266 • 172 b'd 1917 • 432 b'e

aValues are means • SD of six r a t s per each age group. Means not followed by the same letter, c,d o r e, are significantly different (P < 0.001) within each sex. Abbreviations as in Table 1. bSignificantly different from corresponding male r a t at P < 0.001.

TABLE 3 Changes in Lipid Composition and in a-Tocopherol and PCOOH Contents of the Heart and Lung of Male Rats with Aging a Rats (mon) Heart 1 7 12 18 Lung 1 7 12 18

Tissue weight (g)

Total lipids (TL) (mg/g tissue)

PC (mg/g tissue) (mg/g TL)

a-Tocopherol (~g/g tissue)

PCOOH (pmol]g tissue)

0.6 1.5 1.5 1.6

• + • •

0.15 0.1 c 0.1 c 0.1 c

23.0 22.1 18.8 23.5

• 1.9b • 1.85 +_ 0.9 c • 6.8

9.6 10.8 10.0 10.0

• • • +

1.8 0.6 0.8 1.4

435 491 527 539

• • • +

70 b 51 68 28 c

13.7 20.4 21.6 21.4

+ • • •

1.9 b 0.5 c 1.9 c 2.1 c

506 434 635 799

_ +_ • +_

152 b'c 395 129 c'd 175 d

0.9 1.8 2.0 2.3

• _ • •

0.15 0.2 c 0.2 c 0.1 d

25.6 24.6 24.0 20.5

+_ 3.6 • 5.0 + 4.9 • 2.6

6.9 5.8 5.6 5.9

• • • •

0.9 0.8 0.4 0.5

292 230 236 268

+• • •

23 b 44 c 46 14

18.3 22.0 18.8 17.9

• • • •

3.1 0.85 2.5 1.3 c

145 125 88 92

+_ 43 + 39 • 40 • 37

aValues are means • SD of six r a t s per each age group. Means not followed by the same letter, b,c o r d, are significantly different (P < 0.0011 within each organ. See Table 1 for abbreviations.

contents of the heart and lung were not age-dependent. PC contents in the heart and lung were constant during the entire feeding period, except that a higher PC content i n h e a r t T L w a s o b s e r v e d f o r t h e 1 8 - m o n t h - o l d r a t s . or Tocopherol content in the heart and lung did not change w i t h age. P C O O H l e v e l s i n t h e h e a r t i n c r e a s e d s l i g h t l y in the 18-month-old rats, but PCOOH levels in the lung did not increase. Molar ratio of PC and PCOOH. T a b l e 4 s h o w s t h e changes in molar ratio of PC and PCOOH in rat tissues w i t h a g i n g . F o r b o t h t h e b r a i n a n d liver, a g e - d e p e n d e n t proportional increases in PCOOH/PC ratios were clearly evident in male and in female rats. The brain PCOOH/PC r a t i o f o r t h e 1 - m o n t h - o l d m a l e r a t s w a s 0.6 X 10 -5 a n d t h a t f o r t h e 1 8 - m o n t h - o l d m a l e r a t s w a s 3.5 X 10 -5. T h e rate of increase in brain PCOOH/PC ratio was essentially the same males and females. The liver PCOOH/PC ratio o f 1 - m o n t h - o l d m a l e r a t s w a s 1.9 • 10 -5 a n d t h a t o f 18m o n t h - o l d m a l e r a t s w a s 19.7 X 10 -5. A s i m i l a r i n c r e a s e w a s o b s e r v e d f o r f e m a l e liver, a l t h o u g h t h e i n c r e a s e i n PCOOH/PC ratio with time for the females was lower than that of the males. The change in PCOOH/PC ratio in the heart did not correlate with aging, and the lung PCOOH/PC ratio showed no change with aging. Fatty acids composition. T a b l e 5 s h o w s t h e c h a n g e s i n fatty acid composition of brain TL and PC following

TABLE 4

Age-Dependent Changes in the Molar Ratio of Phosphatidyleholine {PC) to Phosphatidylcholine Hydroperoxide (PCOOH} in Rat Tissues a PCOOH/PC (105 • ratio) b

Rats {man) Male 1 7 12 18 Female 1 7 18

Brain 0.6 1.0 2.5 3.5

• • • •

0.1 d 0.1 e 0.2 f 0.4 g

0.8 • 0.1 d 1.8 • 0.1 e 3.0 -- 0.2 f

Liver 1.9 4.5 11.8 19.7

• • • •

0.2 d 0.7 e 0.7 f 2.8 g

3.0 • 0.2 d 7.1 • 0.8 e 11.3 • 0.5 f

Heart 4.0 3.0 4.8 6.1

• • •

0.5 d 0.1 e 0.4 d 0.5 f

n.d. c n.d. n.d.

Lung 1.6 1.7 1.3 1.2

• 0.2 • 0.2 +_ 0.2 • 0.2 n.d. n.d. n.d.

aValues are means • SD of six r a t s per each age group. Means not followed by the same letter, d,e,f o r g, are significantly different (P < 0.001) within each sex. bMolor ratio was calculated for 1-palmitoyl-2-arachidonoyl PC and its monohydroperoxide. CNot determined. a g i n g i n m a l e r a t s . I n t h e T L , o l e i c a c i d (18:1n-9), eicos e n o i c a c i d (20:1n-9) a n d d o c o s a t e t r a e n o i c a c i d (22:4n-6) levels in the 7-18-month-old rats were significantly higher than those in the 1-month-old rats. Docosahexaenoic LIPIDS, Vol. 28, no. 9 (1993)

792

T. MIYAZAWA E T AL. TABLE

5

C h a n g e s in F a t t y A c i d C o m p o s i t i o n of B r a i n T o t a l Lipids (TL) a n d P h o s p h a t i d y l c h o l i n e s (PC) of M a l e R a t s

with Aginga Rats 16:0

(mon)

1 7 12 18

23.9 22.6 23.5 21.7

+ +_ + +_

0.8 1.3 1.4 1.3

18:0 21.8 18.9 19.3 19.6

+ 1.1 c __. 0 . 9 d

+ 1.1 _+ 1.2

Fatty acid (wt%) b 20:1n-9 20:4n-6 TL 0.8 c 0.6 +- 0.1 r 10.0 + 1.3

18:1n-9 19.0 23.9 24.4 25.1

+

--I- 0 . 8 d

_+ 1.1 d ----. 1 . 2 d

1.6 ___ 0 . 2 d 2.0 + 0 . 2 d 2.4 +_ 0.5 d

9.5 + 0.5 9.2 -- 0.9 9.6 + 0.7

22:4n-6 2.2 +__0.2c 2.8 +__ 0 . 2 d 3.1 4"- 0 . 2 d'e 3.2 +- 0 . 1 e

22:6n-3 11.3 + 0.5 c 10.0 _ 0 . 2 d 7.1 + 0.8 e 7.0 + 0 . 7 e

PC 56.7 _ 2.1 c 18.6 _+ 1.3c 11.2 + 0.9c 0.6 + 0.1 c 2.1 -+ 0.3c trace 0.5 __+0.1 c 7 51.1 -!-_1.2 d 16.7 -- 0.9c 15.3 9 1.0 d 1.4 +-- 0.2 d 1.6 -----0.2c trace 0.5 ___0.1 c 12 46.0 +_- 2.4e 13.3 + 0.8d 25.1 • 1.1e 1.4 + 0.2 d 3.0 +---0.2 d trace 1.1 +_ 0.3 d 18 42.1 -!-_1.8e 13.7 ___0.7 d 26.8 "4" 1.2e 1.6 ----. 0.1 d 3.1 +__0.2 d trace 1.0 ___0.3 d aValues are means + SD of six rats per each age group. Means not followed by the same letter, c,d or e, are significantly different (P < 0.001). bIndividual fatty acids are designated by the number of carbon atoms and the number of double bonds, n represents the position of the first double bond from the methyl end. 1

acid (DHA, 22:6n-3) content was lower in 12- and 18month-old rats t h a n in 1- and 7-month-old rats. Saturated f a t t y acid (16:0, 18:0) content remained c o n s t a n t during the 18-mon experimental period. In brain, PC decreased in s a t u r a t e d f a t t y acids (16:0, 18:0) and increased in uns a t u r a t e d f a t t y acids (18:1, 20:1, 20:4, 22:6) with aging. DISCUSSION In the present study, we have demonstrated the accumulation of P C O O H in the brain and in the liver of rats with aging by use of a C L - H P L C m e t h o d for hydroperoxide determination. Such an age-dependent increase in P C O O H was not observed in lung and heart. We also showed t h a t PCOOH was present in all the internal organs we examined at levels of 100 to 2500 pmol per g r a m of tissue (Tables 1-4). These values are m u c h lower t h a n previous d a t a reported for lipid peroxide levels (hundreds of n a n o m o l e s p e r g r a m t i s s u e ) b a s e d on T B A m e a s u r e m e n t s (5-11). I n previous studies, age-related accumulation of lipid peroxides as measured by T B A assay was reported to be 2 or 3 times higher in r a t brain (5,8,9,11,15) and liver (8,9,11,15), and 1.2 times higher in r a t liver lipids b a s e d on conjugated diene m e a s u r e m e n t s (15). In the present study, it was shown t h a t the age-related formation of P C O O H in brain (3.5-4.4 times) and liver (4.7-9.3 times) was more pronounced t h a n previously observed (5,8,9,15). F r o m the evidence provided here b a s e d on d a t a obtained on aging rodents, m e m b r a n e phospholipids in brain and liver appear more susceptible to hydroperoxidation t h a n the lipids in heart and lung. We recently reported the presence of phosphatidylethanolamine hydroperoxide (PEOOH) in addition to P C O O H in rat brain and liver (20). In r a t brain, P E O O H content was generally higher t h a n P C O O H levels. Phosp h a t i d y l e t h a n o l a m i n e in brain and liver is also richer in u n s a t u r a t e d f a t t y acids t h a n is PC; hence, the present findings are not surprising. Liver phospholipid hydroperoxide content is affected by dietary supplementation, e.g., with fish oil, which increases liver P C O O H and P E O O H levels (20). LIPIDS, Vol. 28, no. 9 (1993)

An age-dependent decrease in phospholipid levels has been reported for rat liver (34), h u m a n brain (35), rat brain and liver microsomes (36,37) and for e r y t h r o c y t e membranes (38). In the present study, a slight decrease in brain PC content (Table 1), as well as a decrease in brain PC cont e n t relative to brain TL, was evident (Table 1). The liver PC content was decreased whether expressed relative to liver weight or to liver TL (Table 2). This PC decrease relative to TL and the changes in phospholipid composition m a y have a significant effect on m e m b r a n e fluidity (38,39) in these organs and thus m i g h t change m e m b r a n e function as well (40). As shown in this study, the organs showing a decrease in PC content were the brain and the liver, and only in these two organs was an age-dependent accumulation of P C O O H observed. The PC decrease m a y have resulted from peroxidative breakdown of PC as well as from changes in phospholipid metabolism t h a t m a y occur during aging. The changes in phospholipid composition also seem to directly correlate with the susceptibility to hydroperoxidation of the m e m b r a n e phospholipids themselves (40,41). The content of a-tocopherol as one of the antioxidants in biomembranes did not decrease in the brain and liver upon aging (Tables 1 and 2), although PCOOH had significantly accumulated in these two organs. I t thus appears t h a t atocopherol cannot effectively function as an antioxidant and inhibit PC hydroperoxidation due to aging in brain or liver. I t could alternatively suggest t h a t higher a-tocopherol levels m a y be required to prevent phospholipid hydroperoxidation in the m e m b r a n e s of brain and liver during aging, as was suggested by Meydani e t al. (42). U p o n aging, an increase in superoxide radical formation (8) and a decrease in superoxide d i s m u t a s e activity, catalase activity (11) and glutathione content (9) have been reported to occur in rat brain and in rat liver. The increase in P C O O H in rat brain and liver with age, as shown h e m should also affect m e m b r a n e association and activation of protein kinase C. I t has been reported t h a t oxidative modification of m e m b r a n e lipids can result in persistent activation of protein kinase C (43,44). The livers of 18-month-old male r a t s showed 10.4-fold higher P C O O H / P C ratios than 1-month-old rats (Table 4).

793

LIPID HYDROPEROXIDATION IN AGED RATS T h i s e n h a n c e d P C O O H / P C m o l a r r a t i o in liver l i p i d s of t h e a g e d r a t s w a s however, s i g n i f i c a n t l y lower t h a n t h a t r e p o r t e d for t h e liver of m i c e (32.3 X 10 -5) fed a h e p a t o carcinogenic choline-deficient diet containing ethionine (24). D H A c o n t e n t d e c r e a s e d in T L of t h e b r a i n w i t h a g i n g . On the other hand, arachidonic acid and DHA content s i g n i f i c a n t l y i n c r e a s e d in P C in a g e d brain. B r a i n P C cont a i n i n g a r a c h i d o n i c a c i d o r D H A w o u l d b e e x p e c t e d to b e s e n s i t i v e t o h y d r o p e r o x i d a t i o n in t h e c o u r s e of a g i n g . I n t h e p r e s e n t s t u d y , we h a v e d e m o n s t r a t e d t h a t PCOOH accumulates as a primary peroxidation product of m e m b r a n e p h o s p h o l i p i d s in b r a i n a n d liver of t h e r a t upon aging. These findings provide important evidence t h a t o x i d a t i v e d a m a g e occurs to m e m b r a n e p h o s p h o l i p i d s in t h e c o u r s e of s e n e s c e n c e a n d a g i n g a s s h o w n in t h i s in vivo s y s t e m .

ACKNOWLEDGMENTS This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan and the Skylark Food Science Institute.

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LIPIDS, Vol. 28, no. 9 (1993)