Jouniul (if jNmrodlmlimi. 1976 Vol. 27. pp. 967-968. Pergamon Press Printed in Great Britain

SHORT COMMUNICATION

The differential incorporation of labelled linoleic, y-linolenic, dihomo-y-linolenic and arachidonic acids into

the developing rat brain

(Rrceived 10 March 1976. Accepted 27 April 1976)

THE BRAIN is a lipid-rich organ containing up to 6006 lipid on a dry-weight basis. Its fatty acid composition is unique: brain phosphoglycerides are rich in the C20 and C22 polyunsaturated acids derived from the dietary essential fatty acids-Iinoleic (18:2 w 6) and a-linolenic acids (18:3w3). In all the species so far studied both the latter acids are present in trace amounts (< 1;; of the total fatty acids) while arachidonic acid (20:4 w 6 ) and docosahexa- enoic acid (22:6 ni 3) constitute respectively up to 147; and 251, of the total fatty acids of ethanolamine phospho- glyceride fractions (CRAWFORD & SINCLAIR, 1972).

In the rat it has been shown that both 20:406 and 2 2 : 6 0 ~ 3 accumulate in the brain lipids of the suckling pups during the active period of brain growth and between 60 to SO", of the adult values of these acids are present in the brain phosphoglycerides at the time of weaning (SIN- CLAIR & CUAWFORD. 1972).

Arachidonic acid is synthesized from linoleic acid via a pathway involving desaturation, chain elongation and desaturation: linoleic acid y-linolenic acid

182- 7-18 :3 - di-homo-7-linolenic acid arachidonic acid

2 0 2 -----+ 20:4

It has been shown that in the rat the in iiuo desaturation of linoleic acid is a rate-limiting step in the synthesis of 20:4w 6 (HASSAM et 01.. 1975). To investigate whether or not the desaturation process had an influence in the incor- poration of fatty acids into brain lipids. suckling rats were given radioactive linoleic acid and its desaturated and chain-elongated metabolites.

MATERIALS AND METHODS

Suckling rats, bred from females maintained on a semi- synthetic diet, were used when they were between 15 and

16 days old. They were dosed with either [l-'4C]linoleic acid (61 mCi/mmol, radiopurity 99",", Radiochemical Centre, Amersham, U.K.). [l-14C]~-linolenic acid (20 niCi/ mmol, radiopurity 99", Daiichi Pure Chemical Co.. Tokyo. Japan). [l-I4C]di homo-y-linolenic acid (57 mCi,' mmol, radiopurity 9904, NEN Chemicals GnibH, West Germany) or [l-14C]arachidonic acid (54 mCi/mmol. radiopurity 990,;. Radiochemical Centre, Amersham. U.K.). Four pups were used for each experiment and between 2 to 4 pCi of each isotope was added to 0.25 ml olive oil and administered orally to unanaeshetized animals. The pups were returned to their mothers and killed 22 h later. Animals were killed by decapitation, the brain quickly removed, washed in ice-cold saline. blotted dry and weighed. Lipids were extracted and the radioactivity in the brain lipid fractions and the fatty acids of the brain phos- phoglyceride fractions were determined by using tech- niques previously described. which include the use of TLC and preparative GLC followed by liquid-scintillation counting (HASSAM ef al., 1975; SINCLAIR. 1975).

RESULTS AND DISCUSSION

Radioactivity from each of the administered isotopes was incorporated into the brain lipid of the suckling rats (Table I ) and the highest incorporation was obtained for [l-'JC]arachidonic acid. Most of the radioactivity ( >8On0) recovered was associated with the phospholipid fraction of the brain lipid. The cholesterol fraction was also labelled to a significant degree; however, thc radioactivity de- creased with the degree of unsaturation of the administered fatty acid (Table 1). Very low radioactivity ( < 3 " , , ) was found in the neutral lipids and the glycolipids, the neutral lipids being the predominant of these 2 fractions.

The incorporation of radioactivity into the fatty acids of the brain phospholipid fraction was as shown in Table 2. The label was present in the administered fatty acid

TARLE 1. THE RECOVERY OF THE ADMINISTERED ISOTOPE FROM BRAIN LIPID AND ITS DISTRIBUTION IN THE LIPID FRACTIONS

Isotope administered

Distribution of the isotope between brain lipid fractions (as of the total activity in the brain lipid) Recovery of isotope from

brain total lipid (as 9," of thc administered dose) Cholesterol Phospholipid

[I-'"] 18:2

[l-'"C] 20:3 [I-"C] 20:4

[1-14C]y-18:3 0.44 k 0.5 0.41 k 0.003 1.02 k 0.29 2.56 5 0.14

15 k 0.8 12 & 0.1 8 _+ 0.4 3 0.1

84 4 0.8 84 0.1 89 & 0.3 97 4 0.5

~ ~~~~~

Values are the mean of 4 animals S.E.M. The cholesterol fractions were not contaminated by diglycerides. 967

968 Short communication

TABLE 2. RADIOACTIVITY RECOVERED FROM THE 106 FATTY ACIDS OF THE BRAIN PHOSPHOGLYCERIDF. FRACTION (EXPRESSED AS PtRCEhiTAGE OF THE ADHlh'lSTERED DOSE)

Brain phospholipid

fattj acid Isotope administered fractions [I-l'C] 18:2 [I-"C]7- 18: 3 [I-'"C] 10:3 [l-'"C] 20:4

Saturated and 0.15 * 0.02 monounsaturated 0.15 f 0.01 0.10 & 0.01 0.21 f 0.02

- - - 18:2w6 0.09 f 0.006 0.02 * 0.001 __ - l8:3 w 6 0.02 k n.cm

2 0 . 3 ~ 0 6 0.01 * 0.002 0.04 * 0.001 0.14 2 0.02 - 20:4,6 0.06 k n.oo.1 0. I6 4 0.004 0.40 f 0.03 2.0 0.03

0.27 +_ 0.03 22: 4 (u 6 0.02 2 0.001 0.02 +_ 0.00 I 0.07 0.005

Values are the mean of 4 animals & S.E.hl

and its metabolites; isotope was also recovered from the saturated and monounsaturated fatty acids

The labelling of cholesterol. the saturated and monoun- saturated fatty acids suggests /&oxidation of' the adminis- tered radioactive fatty acids to labelled acetyl-CoA and the reutilisation of the acetate for the synthesis of both the cholesterol and fatty acids. Examination of the lipids of the h e r and plasma show that there is no label in the cholesterol and the saturated and monounsaturated fatty acids (HASSAM er al., 1975: SINCLAIR. 1975). The results of this experiment suggest that the brain is capable of degrading fatty acids by P-oxidation and also synthesiz- ing them via acetyl CoA.

Radioactivity was present in the brain phospholipid polyunsaturated fatty acids both as the administered acid and its metabolites (Table 21. In the pups dosed with [ I-14C]linoleic acid. a lesser percent of the administered dose appeared in 20:4(!~ 6 compared with pups adminis- tered with either [I-'"C]y-18:3. [I-'"C] 20:3 or [l-'"C] 20:4 itself. The highest incorporation of radioactivity into brain phospholipid 20:4 (c) 6 was from administered [I-'"C] 20:4 and the order of incorporation was [I-'"C] 20:4 > [l-14C] 20:3 z [l-'"CJ;.-18:3 > [l-'*C] 18:2. The low incorporation of [I-'*C] 18:2 into 20:4(06 could be explained by the slow synthesis of arachidonic acid fron linoleic acid (HASSAM rt a/ . , 1975). The results demonstrate that the metabolites of linoleic acid are more efficient diet- ary precursors of brain arachidonic acid than is the parent fatty acid.

Since dietary y-lX:3.20:3 (1 ) 6 and 20:4 (O 6 are incorpor- ated into the brain structural lipids more efficiently than their precursor linoleic acid, it is interesting to speculate on the possible relevance to the suckling pup, of the avail- ability of the dietary metabolites of linoleic acid at the time when arachidonic acid is actively accumulated in the brain. The rate of accumulation of arachidonic acid in the brains of the suckling pups would be limited by the rate of desaturation of linoleic acid to arachidonic acid. It has been suggested that low levels of arachidonic acid in the brain structural lipids could account for the higher suscep- tibility of essential fatty acid-deficient rats to allergic ence-

encephalomyelitis (CLAL'SEN & MOLLER, 1967) and may affect the activity of the membrane and membrane-bound enzymes (SLx & SUN, 1974; SUN et ul., 1974). Rat milk normally contains both linoleic acid and its longer-chain metabolites (CRAWFOKD & SINCLAIR. 1972; SINCLAIR, 1974). However. the levels of the metabolites in the milk of lactating rats is influenced by the essential fatty acid intake during lactation (SINCLAIR. 1974; HASSAM. 1976) and hence the levels and type of fatty acid in the diet would influence the availability of arachidonic acid during the period of brain growth.

Acliriowlerlgemenr-We are grateful to the Action For Research into Multiple Sclerosis Ltd. (ARMS) for the financial support.

Deparrmenr of' Biochemistry. Ni@eld Institute of Cotnparatiw

The, Zoologictrl Societj of London. Regent's Park, London. N W 1 4R 1.1 U . K .

A. G. HASSAM M. A. CRAWFORD

Medicine.

REFERENCES

CLAUSES J. & MOLLER J. (1967) Acttr neiirol. s c a d . 43. 375-388.

CRAWFORD M. A. & SINCLAIR A. J. (1972) in Lipids. i2.lalnu- trition crud the Dewlopirzy Bruin pp. 267-287. CIBA Found. Symps. Associated Scientific Publ., Amsterdam.

HASSAM A. G. (1976) Nictr. Mettrh. In Press. HASSAM A. G., SINCLAIR A. J. & CRAWFORD M. A. (1975)

SINCLAIR A. J. (1974) Lipids 9, 809-813. SINCLAIR A. J. (1975) Lipids 10, 175-184. SINCLAIR A. J. & CRAWFORD M. A. (1972) J . Nrurochetn.

SUN G. Y. & SUN A. Y. (1974) J . Neurochem. 22. 15-18. SL~N G. Y.. Go J. & SUN A. Y. (1974) Lipids 9, 450-454.

Lipids 10. 417-420.

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