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The long chain metabolites of linoleic and linolenic acids in liver and brain in herbivores and carnivores
Comp. Biochem. Physiol., 1976, Vol. 54B, pp. 395 to 401. Pergamon Press. Printed #1 Great Britain
THE LONG CHAIN METABOLITES OF LINOLEIC AND LINOLENIC ACIDS IN LIVER AND BRAIN IN HERBIVORES AND CARNIVORES M. A. CRAWFORD, N. M. CASPERD AND A. J. SINCLAIR Department of Biochemistry, Nuffield Institute of Comparative Medicine, Zoological Society of London, Regents Park, London NWI 4RY, England
(Received 14 August 1975) Abstract--l. The parent vegetable polyenoic acids and their long-chain animal metabolites have been studied in liver and brain phospholipids in a herbivorous mammal (Kob) and a carnivore (Hyaena) of similar body size, but contrasting developments of the nervous system. 2. The fatty acid patterns of the phospholipids were found to be tissue and species specific. 3. The major metabolite of linolenic acid was docosapentaenoate (C22:5,n-3) in the Kob liver, but docosahexaenoate (C22:6,n-3)in the Hyaena. Brain grey matter of both species contained the hexaenoate. 4. Comparative studies from 30 different species demonstrated that the brain ethanolamine phosphoglycerides (EPG) had a relatively constant fatty acid composition despite wide variations in the fatty acid composition of liver EPG.
INTRODUCTION
The phosphoglycerides from brain grey matter of m a m m a l s are rich in long-chain metabolites of linoleic a n d linolenic acids (man: O'Brien et al., 1964; rat: Kishimoto et al., 1969; whales a n d dolphin: Lesch, 1969; guinea pig: Crawford & Sinclair, 1972; squirrel monkey: Sun & Sun, 1972). Linoleic (C 18: 2,n-6) a n d linolenic (C 18:3,n-3) acids c a n n o t be made by animals (Holman, 1968) b u t are found in a b u n d a n c e in plant leaves a n d seeds (Hitchcock & Nichols, 1971). These C18 polyenoic fatty acids are partially chain-elongated a n d desaturated by animals to C20 a n d C22 fatty acids with 4, 5 and 6 double b o n d s (Mead, 1968; Klenk, 1972). Hence, the distribution of polyenoic fatty acid types in plant and animal cells is very different and there is a correspondingly wide variation in the dietary polyenoic fatty acids of different species (Crawford & Sinclair, 1972). We wish to report a remarkable constancy of the fatty acids of the brain grey matter ethanolamine phosphoglycerides despite a wide variation in the corresponding fractions from liver. METHODS
Animal species
Lipid extraction Samples of liver, semi-tendinosus muscle and a sample of the brain were stored at -20°C until analysed. The grey matter was separated from white matter in the region of the motor cortex. Weighed samples of frozen tissues were homogenised (M.S.E. Top drive) under nitrogen in twice the volume of cold (+4°C) chloroform-methanol (2:1) containing 10 mg/1 of recrystallised 2:6 di-t-butyl-pcresol as an anti-oxidant. The tissue samples were extracted twice and the combined extracts washed with 0.9% saline and evaporated to dryness at reduced pressure.
Phospholipid separation Aliquots of the lipid extracts were separated into the phospholipid fractions by thin-layer chromatography (TLC) on silica gel G using chloroform-methanol-7 N ammonia (65:30:4.5) as the solvent system. The fractions were located by dichlorofluoroscein (0.2% in methanol) and the use of adjacent standards on the same plate. The EPG and choline phosphoglycerides (CPG) bands were scraped from the thin-layer plates and transmethylated with 5% H2SO 4 in methanol at 70°C for 3 hr. The EPG and CPG fractions of muscle and brain are mixtures of diacyl-phosphoglycerides and plasmalogens (alkenyl, acylphosphoglycerides); acidic methanolysis of such a mixture produces dimethyl acetals (DMA) from the fatty aldehydes (Sun & Horrocks, 1968) and fatty acid methyl esters from the fatty acids. The DMA were included in the analysis.
Fatty acid identification The Uganda Kob (Adenata Kob) and the spotted Hyaena (Crocuta crocuta) were chosen for this study on The methyl esters and DMA were separated using a Pye the grounds of availability and similarity in adult body Gas Chromatogram (Series 104, W. G. Pye & Co. Ltd.) size. Six male adults and one female of each species and an $6 Research Chromatograph (Fisons Ltd.), both between 4 and 6 years old were selected from the Ishasha incorporating flame ionisation detectors. In both instruReserve on the borders of the Queen Elizabeth National ments, 5 ft glass columns were used with the packings of Park and from the Tonia/Kaiso flats, Uganda. The animals 10% polyethylene glycol adipate (PEGA) or 10~, Apiezon were shot with a rifle and tissues were frozen using solid M, both on 100-200 mesh celite and run routinely at CO2 within 4 min after killing. Body weights (without sto184.5 and 210°C, respectively, with an argon flow rate of mach contents) were 73 ___5.2kg for the Kob and 52 ml/min. A copolymer of ethylene glycol succinate with 64 + 4.8 kg for the Hyaena (mean + S.E.). Tissues of other methyl silicone at 10% on 100-120 mesh gas chrom P wild mammals were obtained from several sources (Table (EGSS-X) was also used under the same conditions at a 5). temperature of 190°C. The relative retention volumes of 395
396
M.A. CRAWFORD,N. M. CASPERDAND A. J. SINCLAIR
unsaturates to saturates is larger on EGSS-X by comparison with PEGA. This difference in performance characteristics enabled overlap ambiguities such as the coincidence of nervonic acid (C24:1) and clupanodonic acid (C22:5,n-3) on PEGA to be resolved; conversely on EGSS-X the eicosaenoic acid can coincide with linolenic acid but separates with a longer retention time on PEGA. The Apiezon column was used to establish carbon chain length of the methyl esters. Argentation chromatography was employed to confirm identity by separation of the methyl esters based on the degree of unsaturation, followed by hydrogenation to establish carbon chain length. The retention time on PEGA and EGSS-X for fatty acid methyl esters with the same carbon chain length increases with increasing degrees of unsaturation and is also dependent on the length of the saturated carbon chain measured from the methyl end. This characteristic enables the positioning of the double bond sequence to be determined (Ackman, 1969). A methylene interrupted sequence of double bonds was assumed when there was agreement of relative retention values with standards on both PEGA and EGSS-X, or with separation factors when standards were not available. Standards of eicosapentaenoic (C20:5,n-3) and arachidonic acid (C20:4,n-6) were obtained from the Hormel Institute. We were given the C22:4,n-6 by Professor D. A. van Dorp (Vlaardingen Research Laboratories, Unilever, Holland), the purity of which had been determined to be better than 98~o. The identity of the C22:5m-3 was established by the use of separation factors and by combined gas liquid chromatography--mass spectroscopy, and C22:4,n-6 by argentation chromatography and confirmed by mass-spectroscopy through the assistance of Associated Electrical Engineers. Docosahexaenoic acid (C22:6,n-3 with double bonds in the 4, 7, 10, 13, 16 and 19 positions) is readily available from cod liver oil which was kindly supplied by British Cod Liver Oils (Hull & Grimsby) Ltd.
Table 1. Phospholipid composition of muscle, liver and brain grey matter from the Kob and Hyaena
Species
ayae~ ~ob
Hyae~ Kob
Tissue
The nomenclature used here to describe the fatty acids follows the convention of Ca:d,n-x, where a refers to the carbon chain length, x the length of the saturated carbon chain from the methyl end and d the number of double bonds. Because of the increasing need to refer to the docosahexaenoic acid with methylene interrupted bonds in positions 4, 7, 10. 13, 16 and 19, it was felt worth introducing a trivial name for this acid and we suggest "Cervonic acid". Similarly, there is a frequent need to refer to the short chain polyenoic acids, linoleie and linolenic acids, as a specific group and separately to their chain elongation and desaturation products with 20 or 22 carbon chain lengths and two to six double bonds. Hence we shall refer to the former group as the short chain polyenoates (SCP) and the latter group as the long chain polyenoates (LCP). The results are expressed as g/100g (i.e. wt ~o) of the total fatty acid and aldehyde within the individual phospholipid fraction. In most instances data have been collected from more than one representative of each species, but in the interest of brevity the data corresponding to one of each species is recorded here except for those on the Kob and Hyaena. In all cases there was close agreement within an individual species suggesting a degree of species specificity. Whilst it was possible to obtain the tissue samples within 0.5-4min after death this was not so with the human samples which were obtained at post-mortem.
Molar Percentage
EPG
PC
Brain
26.1
35
34
14
13
Brain
25.3
55
36
ii
16
Liver
16
29
45
5.2
9.4
Liver
12
27
51
7.6
8.I
PS
SPH
there were quite marked species variations in the acyl group of liver and muscle phospholipids. The brain phospholipids were remarkably constant (Tables 2-4). The K o b liver had more C18:2,n-6 and C18:3,n-3 in the phospholipids compared with the Hyaena but arachidonic and the C22 polyenoates were present in higher proportions in the Hyaena liver than in the K o b (Table 2). In the K o b liver phospholipids, the major long chain derivative of linolenic acid was clupanodonic acid (C22:5,n-3), but cervonic acid (C22:6,n-3) in the Hyaena. This difference is also observed for the muscle phospholipids from these two species (Table 3). The analysis of one liver from a female of each species has shown a distribution of Table 2. The proportions of linoleic, linolenic acids and their long-chain derivatives in the liver phospholipids from the Kob and Hyaena
Fatty Acids
T er minoloy y
Total phospholipid g/lO0 g dry weight
Liver EPG Kob
10:z,=-6 20:2,n-6 20:5,~-6 20:4,n-6 22:4,n-6
~6,5,~-3 20,5,~-3 22:5,n-3 22:6,~-5
Liver CFG
Hyaena
Kob
Hyaena
8.2+0.5 0.5+0.06 1 .i+o .o6 12 +-0.4 I.C+0.1
5.0+0.1 0.6_+0.07 1.o*_o.o5 15 +0.5 O.}+0.o1
9.1+0.8 0.2_+0.01 o.6+O.Ol 6.0+_0.07 1.0+0,06
4.3+0.2 0.6+0.01 5.1+0.06 12 +0.4 0.6+o.01
4.4_+0.5 3.8+_o.i ii +o.i i. 6+-o.03
2.~0.09 0.4+-O.l 4.2+0.1 14 -+0.6
4.9*_0.5 4.8_+0.5 8.6z_o°i 1.1+o.2
2.7+_o.o8 o.smO°Ol 3.0+0.05 7,6+0.2
Results expressed as g fatty acid/100 g total fatty acids. Mean + S.E.M. for 6 samples of liver from each species. Table 3. The proportions of linoleic, linolenic acids and their long-chain derivatives in the muscle phospholipids from the Kob and Hyaena Fatty Acids
}@~scle E ~ Kob
10:2,~-6 2o: 5,n-6
20,4,~-6 18:3,n-3 20:5,n- 3 22:5,n-5 22:6,n-5
]~scle CPG Hyaena
Kob
Hyaena
II +--0.6 0.3+-0.05 10 +-0.8
3.1+-0.6 1.6+-0.1 15 +0°4
13+_1.6 0.4_+0.03 7.6+-0.1
4.3+-0.7 1.1+0.07 9.2+_0.1
3.2±0°4 2.1~0.0~ 7.2+-0.4 0.8-+0o01
O.9±o.05 1.0~0.04 5~0-+0.6 6.1-+0.3
3.9±0°7 1.6+-0.08 5.6+0.O9 0.4-+u°02
1.8+-0.1 i.i+-0.05 4.8-+0.2 4~7+-0.7
RESULTS
The content of total phospholipid and of the major phospholipid fractions in liver and brain from the K o b and Hyaena were similar (Table 1). However,
Results are expressed as g fatty acids/100g total fatty acids and aldehydes (see Methods). Mean _+ S.E.M. for 6 samples of muscle from each species.
Long chain metabolites in herbivores and carnivores Table 4. The proportions of linoleic, linolenic acids and their long-chain derivatives in the brain grey matter phospholipids from the Kob and Hyaena Fatty
Table 5. Sources of the material used in this study Groups
Source
CPG
EPG
Acids Kob
Hyamm
Kob
I~erm
18:2,n-6 20:2,n-6 20:5,n-6 20:4,n-6 22~4,n-6 22:5wn-6
0.6 0.~ 0.6 12 8.0 0.5
0.I 0.2 0.3 16 5.9 i.O
0.6 0.~ 0.5 8.6 1.5 0.9
0.4 0.2 5.8 2.4 1.0
18:5,n-3 20:5pn-5 22:5,n-3 22:6,n-5
0.4 1.5 1.9 25
0.2 0.9 1.6 24
0.2 I.i 3.2 5.2
0.2 0.7 1.8 7.5
_
397
Results expressed as g fatty acids/100 g fatty acids and aldehydes (see Methods). One brain sample from each species analysed in triplicate and the results are presented as the mean.
the fatty acids similar to that seen in the male animals. The brain grey matter phsopholipids in both species (Table 4) were characterised by the presence of long-chain polyenoic acids (C20 and C22), but only trace amounts of the parent C18 polyenoic acids. The EPG fraction from grey matter of both species was of particular interest as the principal fatty acid was cervonic acid, which was not the case in the Kob liver lipids. In the liver, muscle and brain from both species, the EPG fraction consistently contained a higher proportion of the long chain (C20 and C22) polyenoic fatty acids by comparison with the CPG fraction (Tables 24). As the EPG fraction of liver and brain grey matter was the richest in the long-chain polyenoic acids we selected this fraction for study in a variety of different mammalian species. The location from which these species were chosen is given in Table 5. From Tables 6 & 7 it can be seen that there were marked differences in the liver EPG but the brain EPG values were similar. In the brain, EPG was rich in LCP in the grey matter, but not in the white matter (M.A.C. unpublished data). As far as possible the samples of brain were taken from the motor cortex and were principally composed of grey matter. However, in small mammals this was not always possible and the data representing EPG from whole brain are indicated in Table 6; the consequent mixture of EPG would tend to dilute the LCP component. It might have been possible to overcome this difficulty by separating brain cells, but in this first analysis it was decided to measure whole tissue in view of the instability of the LCP. In the brain EPG the major polyenoic constituents were the LCP which appeared in the proportions of C22:6 > C20:4 > C22:4 > C22:5,n-6 and n-3 > C20:5. The percentage of C22:6,n-3, C20:4,n-6 and C22:4,n-6 averaged 21.4 + 0.82, 11.5 + 0.46 and 5.72 _+ 0.3 in data for the 28 species in Table 6 (mean Yo +- S.E.M.). By contrast, the parent C18 fatty acids of vegetable origin, linoleic acid (C18:2,n-6) and lino-
Small I,~ma I s Rat (Rattus sp.) Guinea-pig (Carla porcellus) Hamster (Cricetus cricetus) Steppe Len~ning (Laguras) Viscacha (Lagostomus maximus) Cuis (Galea musteloides) Casaragua (Proechimys guairae) Rock Hyrax (Procavia capensis) Fruit-eating Bat (Megachiropterus) Large Non-ruminants Wart-hog (Phacochoerus aethiopicus) Elephant (Loxodonta africana) Horse (Equus caballus) Zebra (Equus burchelli) Large Ruminants Topi (Damsliscus korrigum) Bu/falo, woodland (Syaoerus caffer) Hartebeest (Alcelaphus buselaphus) Elana (~uro~-agu~
oryx)
Giraffe (Giraffa camelopardalis) Red ~eer (Cervus el~phus) Ox (Bos taurus)
Laboratory, N.I.C.M.
Kilimanjaro, Tanzania Makerere, Uganda Ishasha, Uganda Newmarket, U.K. South Karamoja, Uganda, and Z.S.L. Ishasha, Uganda South Karamoja, Uganda ,, ,, Z.S.L. London, U.K.
Carnivores Cat (Fells catus) Civet (Viverra civette) Leopard (Panthera pardus) Lion (Panthera leo) Canadian Timber Wolf (Canis lupus)
Laboratory, N.I.C.M. Bulemezi, Uganda Ishasha, Uganda Z.S.L.
Primates Marmoset (Leontideus rosalia) Patas Monkey (E~ythrocebus pates) Vervet Monkey (Cercopithecus aethiops) mamma (~omo s s p i ~ )
Laboratory, N.I.C.M. N.I.C.M, and Madl, N. Uganda (origin, Uganda) London
Marine Mammals Bottlenosed Dolphin (Tursiops truncatus)
Z.S.L.
lenic acid (C18:3,n-3) had averages of 0.93 + 0.1 and 0.26 + 0.04 respectively. That is, the brain was characterised by a high LCP and low SCP content. This degree of uniformity of brain EPG fatty acid profile was not found in the liver EPG. For example, the mean linoleic and linolenic acid proportion in the liver EPG of small mammals was 13.1 + 1.8~o and 1.3 + 0.54~o which is many times greater than that which was found in the brain. The arachidonic acid of 10.8 + 1.5~o and the cervonic acid of 19.5 + 2.5~o were similar to the levels found in the brain but the docosatetraenoic acid (C22:4,n-6) was much lower in the liver EPG at 0.5~o ___0.12~o. The guinea-pig reaches an adult body weight of about twice that of the rat. In the case of the rats and guinea-pigs studied here, both were fed the same diet, yet the EPG of the guinea-pig contained significantly less LCP but more SCP than was found in the rat liver EPG. The contrast between brain and liver EPG was even greater in the case of the large non-ruminants or what might loosely be referred to as herbivores, except that the warthog is in principle omnivorous even if, in our experience, it is predominantly a herbivore in practice. In this group the linoleic acid proportion was remarkably high. Although this group only contains four species the analyses represent a total of 15 animals and there was close agreement in the data obtained within a given species. The liver EPG linoleate of the five warthog averaged 23.2 + 1.6~o and the four elephant 11.0 + 1.8~o. The mean linoleate of all four non-ruminant species was 24°/,. The zebra was of special interest because of the
398
M.A. CRAWFORD,N. M. CASPERDAND A. J. SINCLAIR Table 6. Brain grey matter EPG polyenoic fatty acids n-6
T
12 12 l0 II
6.5 7.1 4.9 7.2
0.8 0.6 0.9 0.4 0.2 0.7
II I0 ll 12 14 14
0.6 0.7 0.8
0.2 o.I
i,2 1.5
0.5 0.2
0.7 O°6
2.1
0.5
0.6
0.6 0.9 0. 9 1°5
25 19 18 25
7.2 6.9 6.3 6. 5 7.0 6.2
o.8 1.2 0.8 I.i 1.2 o.9
o.7 0.7 0.5 I,I 0.8 0.4
0.6 0.8 0.6 0,5 0.4 o.7
Oo7 0.2 1.2 0.4 0,5 0.5
17 25 22 16 21 24
13 14 14
6.7 5.9 7.1
I.i 1.5 1.o
0.} O.4
0.8 0.8 0.5
0.8 0.5 0.7
27 26 22
0.5
9
5.0
0.5
o,4
16
0.I O.1 0.5 1.0
ll 6.8 14 14
4.7 5.3 6.8 8.O
1.6 1.2 0.5 2.2
0.i 0.8 0.9
0.5 0.6 0.5 1.6
29 19 21 25
0.i
6.9
5.6
0.2
0.8
2.5
27
Lar6e Non-Ruminants Wart-hog 1 Horse 4 Zebra 2 Elephant 3
0.9 1.0 O.1 i.I
0°l 0.4 0.1 0.2
0°2 o.7 0.5 0.8
i 1 6 1 5 1
1.5 0.8 1.2
0.5 0.3 0.6
2.5
0.5
1.7 1.4
0.2 0.2
2 1 1
O°9 1.2 ~ 0.7
0.2 0.3
Primates Marmoset Vervet
Patas Man Marine b~m~ Is Dolphin
I 1 1 4 5
0.8 0.i 0.6 O.5
2
0.7
0.1 0.6 0.2 O.7
i
22:5 0.5 0.6 0.2 0.7 i.i 1.5 0.6 1.4
0.7 0.i 0.3 0.5
Carnivores Cat Civet Leopard Canadian Timber Wolf
-
20:5 0.2 0.6 0.4 0.4 0.4 0.5 0.9 o°5
20:5 0.5 i.i 0.5 1.O 0.9 O. 5 0.4 0.6
Ox Buffalo Eland Giraffe
n-5
18:3
20:2 0.i 0.2
Red Deer
i
22:5 0.6 2.9 0.2 1.5 0.7 1.2 0.6 2.5 0.5
18:2 0.5 1.6 1.5 1.5 1.4 0.5 0.6 0.9 0.5
Larl~e Ruminants Hartebeest
,
22:4 5.1 7.9 4.2 4.5 6,1 4.7 2.9 5.2 1.1
Small Mammals n Rat i0 Guinea Pig 10 Hamster 2 Steppe Le~mning 6 Viscacha l Cuis i Casaragua i Fruit-eating bat 2 Vole 1
20:4 II 17 9.6 i0 12 8.5 9 •7 15 ii
0.5
0.i
22:6 21" 19 22 29* 16 13" 15" 21 2O*
A similar difference between liver and brain EPG was seen in the large ruminants in which the liver fatty acids were characterised by a high SCP and low LCP content. In the eight species studied the mean linoleate and linolenate proportions were 10.2 ___ 1.5% and 2.8% respectively. Liver arachidonic acid levels
remarkably high linoleate (47 and 45%) in both specimens studied. Another striking difference between the liver and brain EPG in this group was the very low proportion of cervonic acid (1.5%) which characteristically constituted about one-fifth to one-quarter of the fatty acids of cortical grey matter EPG.
Table 7. Liver EPG polyenoic fatty acids r
o.1 0.2 0.I 1.3
o.6 1.8 0.3 4.5
12 6,2 4.2 i0
o.5 o.5 o.1 0.4
0.i 0.4 0.I 0.i
5.5 0.9 1.6 2.8
5.7 1.0 0.5 5.8
5.o 2.8 1.0 ll
1.6 5.6 0,2 0.6
Red deer
5 4 2 8 4 4 4 2
14 ii 5.1 4.0 9.7 8.5 12 17
0.2 0.2 0.5 0.9 0.2 0.2 0°2 0.9
0.3 0.4 0.5 1,2 0.2 0.5 0. 5 0.9
9.2 14 11 16 13 14 12 16
0.5 0.7 0oi 2. 5 0.5 0.6 i,i 1.9
0.5 0.5 0.I 0.6 o.l 0.2 0.8 0.8
4.0 5.3 3.5 1.0 3.6 2.8 2.1 1.9
4.1 2.9 7.8 5.1 2.6 1.9 2.1 2.7
9.0 ll 8.5 II 12 7.8 9.~ 8.1
0.8 0.9 0.5 2,5 1.8 6.4 0.8 4°8
Ox (domestic) Buffalo Eland Giraffe Camel Carnivores ~ t i c ) Civet Leopard Li on
2 3 2 I
5.5 5.4 5.8 2.9
0.2 o.5 0.I 0.5
1.4 0.5 O.9 1.7
18 15 15 16
1.5 5.6 2.4 5.6
1.0 1.1 0.7 1.4
1.5 2.0 1.6 0.2
0.9 2.0 O.4 1.5
0.5 0.8 2.O 7.7
22 20 18 9.4
Pri~tes Marmoset Vervet
Patas Human (adult) Foetus
I 2 4 3 i
i0 9.2 9.0 6.3 Ooi
o. 5 1.3 0.5 0.8 o.75
o,6 o.8 3.1 4.2 1.54
23 17 18 16 5o
0. 7 O.5 0.8 5.5 5.4
i,3 o.4 o.5 1.5 1.4
I,i 0.5 2.3 0.2 0.5
o.4 5.2 2.5 1.6 0.5
o.5 2.6 4.8 4.o 0.5
8.2 5.9 8.9 7.7 15
b~rine ~rm~al Dolphin
2
1.7
o.2
13
o.9
o.9
O.1
8.2
1.5
ll
-
20:4 21 11 13 7.6 ll 14 4.1 9.5 ii 5.5
22:4 0.6 1.3
22:5 0.4 0.9
0.5
0.5 0.5 0.2 0.4 0.2
n-3
22 17 47 lO
11
20:2 o.i O.1
,
o.I 0.3 0.5 0.2 o.i 0.2
Large Non-Ruminants Wart-hog 5 Horse(domestic) 4 Zebra 2 Elephant 4
18:2 6.0 25
,
16 20 16 8.5 16 8 7.2
n I0 i0 Hamster 2 Steppe Lemming 6 Viscacha i Rock ~ r a x i Cuis i Casaragtm i Fruit-eating bat 2 Vole i
Large Ru~nants Topl Hartebeest
n-6
20:3 0.5 0.5 0.5 0.5 0.7 1.O 0.2 0.l I.i 0.2
Small M~mmals Rat Guinea Pig
18:3 o.l 5.4
20:5 1.5 2.2
22:5 5.5 2.8
0.5
0.3
o.7
1.2
0.9 0.3 0.2 0.2 0.05
0.7 0.6 3.2 0.3 0.2 0.6 1.8
1.7 1.6 0.6 1.7 1.9 1.4 1.0
i.o 1.1 0.9 2.2 0.5 5.4 2.2
22:6 21 10 23 21
22 21
52 15 5,9 26
Long chain metabolites in herbivores and carnivores
399
were similar to the brain at 13.1 + 0.84~o but the cervonic acid proportion was only 2.3 + 0.78~o. It was of interest that in both the large herbivore groups the clupanodonic acid (C22:5,n-3) was greater than the cervonic acid (C22:6,n-3). In the large ruminants the clupanodonic acid averaged 9.6 + 0.56~o. The carnivore liver EPG was characterised by a significantly lower proportion of SCP and a significantly higher proportion of LCP in comparison with the herbivores. This difference in profile is the same as was found for the direct comparison between the Kob and Hyaena. In summary, this comparison of the EPG of liver from large herbivorous mammals established them to be relatively rich in SCP whilst the carnivore livers were relatively rich in LCP but not SCP. The profile found in the primates included both SCP and LCP. Despite this diversity of liver EPG the brain EPG was similar in all species. The opportunity arose to study two dolphins. Whilst the land food chain provides both the n-6 and n-3 series of acids the marine food chain is predominantly concerned with n-3 fatty acids (Ackman, 1964). Because of this extreme difference between the food chemistry on land and in the sea it was felt that the dolphin brain might be different. It was therefore surprising to find that not only was the brain EPG similar to that of land mammals, but also the liver EPG of this species accumulated both series of acids, suggesting either a specialised food selection pattern or an ability to concentrate the n-6 series of acids from the small amounts available in the principal fish species. In Table 8 the total lipid content of liver and brain cortex grey matter is expressed on a dry weight basis. The high proportion of the lipid in the brain illustrates that lipid accounts for the highest proportion of brain solids.
interaction of lipid or polyhydric alcohols and trace minerals or complex prosthetic groups with the protein molecule. However, it is generally believed that the functional unit is primarily dependent on the initial code for the amino-acids and the unique differences between the amino-acids, although the chemical differences between many of the individual aminoacids is not great. For example, the essential aminoacid leucine is distinguished from the non-essential amino-acid alanine only by two methyl groups; leucine and isoleucine have identical empirical formulas yet are both referred to as separate and non-interconvertible essential amino-acids. Similarly, there are both essential and non-essential fatty acids in the structural lipids and the distinction between the chemical structures of the fatty acids does not appear to be great, at least on paper. Oleic (C 18 : 1,n-9) is distinguished from linoleate (C 18 :2,n-6) by one double bond and linolenate by an additional double bond (C18:3,n-3); however, the positioning of these and the number of double bonds in the fatty acid not only affect their physical properties (e.g. melting point, Chapman, 1972) but also their biological properties, for animal systems are unable to insert double bonds in the n-6 and n-3 positions. This means that animals can synthesise oleic but not linoleic nor linolenic acids and cannot convert linoleic to linolenic acid (Mead, 1968). Linoleic and linolenic acids occur in plants and there is evidence that they both exhibit "essential fatty acid" properties (Houtsmuller, 1973), Furthermore, the LCP have different activities to the SCP. For example, arachidonic and cervonic acids are incorporated some 10~0-times faster into the developing brain by comparison with linoleic and linolenic acids (Sinclair & Crawford, 1972; Sinclair, 1975). Both linoleic and linolenic acids are metabolised in the liver to derivatives in which the chain has been elongated at the carboxyl end of the molecule. The DISCUSSION liver enzyme systems are also capable of introducing additional double bonds between the carboxyl and The two principal building classes of biochemicals the existing methylene interrupted sequence. As a in the cells of soft tissues in advanced forms of life result the liver makes available two families of highly are the proteins and lipids. Protein is quantitatively unsaturated fatty acids, i.e.: the most important in muscle whilst the lipids are quantitatively the most important in the brain and 18:2,n-6 ~ 18:3,n-6 ~ 20:3,n-6 --~ 20:4,n-6 nervous system. Proteins are mainly built from 20 22:4,n-6 ~ 22:5,n-6 amino-acids, eight of which are usually considered 18:3,n-3 ~ 18:4,n-3 --~ 20:4,n-3 --~ 20:5.n-3 essential to animals, that is, they cannot be synthe22:5,17-3 ---, 22:6,n-3 sised by animals or cannot be synthesised fast enought to meet the demands of cell growth. In the structural lipids these polyenoic fatty acids The complexity of the protein system is dependent are found in the /3 position of the phospholipid. In on the sequence of the amino-acids. Further elabo- general all the main polyenoic acids underlined are ration of the structure is achieved by the folding and found in the structural lipids of animals although interaction of the polypeptide chains and finally the within any given animal species a tissue specific patfunctional unit of the cell is dependent on a further tern seems to exist (e.g. adrenal cholesteryl esters: Table 8. Mean lipid content of brain cortex and liver per 100 g dry wt LIV~
No.
Mean
21"
22.9
BRAn~ Grey Matter
+
S.E.
No.
2.2
20
Mean 51-9
S.E. +
1.36
* Excludes small mammals where brains were not separated into white and grey.
Ostwald et al., 1964; Carney et al., 1971 and testis lipids: Bieri & Prival, 1965). Our results confirm that in the brain neither linoleic nor linolenic acids appear in significant amounts and in a wide range of advanced mammalian species the brain polyenoic fatty acids were restricted to the LCP of C20 and C22 carbon chain length: regardless of species, food selection pattern or liver tissue lipids, the pattern of polyenoic acids found in the brain was substantially the same. This fact has some interesting implications;
400
M.A. CRAWFORD,N. M. CASPERDAND A. J. SINCLAIR
the LCP are not commonly found in plant lipids and therelore the production of a given structural lipid profile of fatty acids is dependent both on the metabolic sequence and on exogenous supply. Both the manner of supply and metabolism demonstrate that the principles of assembly of lipids and proteins during cell growth are different in a fundamental respect. In protein assembly, individual amino acids are incorporated in the sequence. In lipid assembly the parent fatty acids have to be metabolised further and both parents and metabolic products are incorporated' into the cell structural lipids. That is, the difference amounts to the use of discrete individuals (amino acids) as opposed to the use of families (fatty acids). A further difference lies in the exogenous source of lipids and amino-acids. Photo and microbial synthesis produces all the amino-acids found in animal proteins; plants provide the parent SCP. This means that there are two exogenous sources for members of the essential fatty acid families found in animal cell structures: plants provide SCP and animal products LCP. If the metabolism of SCP to L C P was rapid in animals, the likelihood of animals accumulating significant amounts of SCP in their tissues would be low. In all animal species we studied, significant amounts of SCP did accumulate in the E P G ; in the cell structural lipids. Indeed, it has been argued that a diversity of fatty acids is an important structural feature of these lipids (Crawford & Sinclair, 1972) and this diversity would not be possible if instant conversion of SCP to L C P did occur. Indeed, from the evidence of the liver lipids presented here, it is plausible that the small, but not necessarily the large animals have the ability to elaborate a complete fatty acid profile. (cf. large ruminants and non-ruminants with small mammals Table 7). It was especially interesting that it was the fastest growing animals and those which reach the greatest adult body weights in which the chain elongation and desaturation process did not appear to reach completion in the liver and muscle tissue. Yet even in these species only the fully desaturated L C P were found in the brain. The fast growing large mammals have the smallest brains in proportion to body weight (Spector, 1956). Both these facts are consistent with the suggestion that the factors concerned with brain growth need not be the same as the factors concerned with body growth; the plausibility of this suggestion is supported by Sacher's (1968) foetal growth analysis, which illustrates wide contrasts in body and brain growth in different species. Also the buffalo, zebra and horse can achieve body weights of about 500-1000 Kg in 3-4 years and have a relative brain to body weight of 0.06%, whereas man only achieves a body wt of 7 0 K g after 18 years of growth and has a brain body wt ratio of 2%. As the brain is predominantly lipid (Table 8) this suggestion might be expressed in biochemical terms by saying that protein synthesis could proceed at a different rate to lipid synthesis. The accumulation of both SCP and LCP in liver structural lipids, the failure of large, fast-growing herbivores to fully desaturate liver and muscle LCP
despite an obvious abundance of the SCP precursors and the progression of the polyenoic acid profile from herbivore to carnivore towards the LCP could be explained by a slow rate of conversion of SCP to LCP, rate-limited by the desaturation step (Hassam et al., 1975). The remarkable specificity of the brain polyenoic acids raises a number of interesting questions pertinent to the evolution of large animals and the obvious disparity between the accumulation of body and brain weight. The biochemical evidence presented here suggests that the long-chain 15olyenoic acids could be a qualitative determinant of brain development in higher animals. Acknowledgements We are grateful to Mrs. Lynne L. Springett, Miss P. Stevens and Mr. G. Williams for skilled technical assistance. We are also pleased to record our appreciation of the assistance given by the Game Departments of Uganda and Tanzania without whom this work would not have been possible. We are indebted to Professors J. Boldingh and D. A. van Dorp of Unilever for technical discussions and for fatty acid standards, and to Unilever Vlaardingen for financial assistance. We are also grateful to Dr. I. Keymer and Mr. D. Jones of the Zoological Society of London for assistance with specimens.
REFERENCES
ACKrC~N R. G. (1969) Gas liquid chromatography of fatty acids and esters, in Methods in Enzymology, Vol. XIV, pp. 329-381. Academic Press, New York. BIERI J. G. & PRIVAL E. L. (1965) Lipid composition of testes from various species. Comp. Biochem. Physiol. 15, 275-282. CARNEYJ. A., SLINGERS. J. & EALKERB. L. (1971) Homo7-1inolenic acid: a major polyunsaturated fatty acid of swine adrenal cholesteryl esters. Lipids 6, 624. CHAPMAN D. (1972) The role of fatty acids in myelin and other important brain structures. In Lipids, Malnutrition and the Developint4 Brain. pp. 31-50. Associated Scientific Publishers, Amsterdam. CRAWFORD M. A. & SINCLAmA. J. (1972) Nutritional influences in the evolution of the mammalian brain. In Lipids, Malnutrition and the Developing Brain. pp. 267 287. Associated Scientific Publishers, Amsterdam. HASSAMA. G., SINCLAIRA. J. & CRAWFORDM. A. (1975) The incorporation of orally fed radioactive 7-1inolenic acid and linoleic acid into liver and brain lipids of suckling rats. Lipids 10. 417-420. HITCHCOCK C. & NICHOLS B. W. (1971) Plant Lipid Biochemistry, pp. 81-93. Academic Press, New York. HOLMANR. T. (1968) Essential fatty acid deficiency. In Progress in the Chemistry o]' Fats and Other Lipids, Vol. IX, pp. 275-348. Pergamon Press, Oxford. HOUVSMOULLERU. M. T. (1973) Differentiation in the biological activity of polyunsaturated fatty acids. In Dietary Lipids and Postnatal Development, pp. 145-155. Raven Press, New York. KISHIMOTOY., AGRANOFFB. W., RADIN N. S. & BURTON R. M. (1969) Comparison of the fatty acids of lipids of subcellular brain fractions. J. Neurochem. 16, 397~404. KLENK E. (1972) The polyenoic fatty acid and the problem of essential fatty acids. In Current Trends in the Biochemistry of Lipids, pp. 41-48. Academic Press, London. LESCH P. (1969) The development of the neutral lipids and fatty acids in the brain of man, whales and dolphins. Clinica Chim. Acta 25, 269 282. MEAD J. F. (1968) The metabolism of the polyunsaturated fatty acids. In Progress in the Chemistry of Fats and Other Lipids, Vol. IX, pp. 159-192.
Long chain metabolites in herbivores and carnivores O'BRIEN J. S., FILLERUP D. L. & MEAD J. F. (1964) Quantification and fatty acid and fatty aldehyde composition of ethanolamine, choline, and serine glycerophosphatides in human cerebral .grey and white matter. J. Lipid Res. 5. 329-338. OSTWALD R., SHANNON A., MILJANICH P. & LYMAN R. L. (1964) Adrenal lipids of cholesterol-fed guinea-pigs and rats. J. Nutr. 82, 443-451. SACHER G. A. (1968) Amin. Zool. 8, 820-821. SINCLAIR A. J. t~ CRAWFORD M. A. (1972) The incorporation of linolenic acid and docosahexaenoic acid into liver and brain lipids of developing rats. FEBS Lett. 26, 127-129.
401
S1NCLAIR A. J. (1975) The incorporation of radioactive polyunsaturated fatty acids into the liver and brain of the developing rat. Lipids 10, 175-184. SUN G. Y. & HORROCKS L. A. (1968) The fatty acid and aldehyde composition of the major phospholipids of mouse brain. Lipids 3, 79-83. SUN G. Y. & SUN A. Y. (1972) Phospholipids and acyl groups of synaptosomal and myelin membranes isolated from the cerebral cortex of squirrel monkey (Saimiri sciureus). Biochim. Biophys. Acta 280. 30(~315.
THE LONG CHAIN METABOLITES OF LINOLEIC AND LINOLENIC ACIDS IN LIVER AND BRAIN IN HERBIVORES AND CARNIVORES M. A. CRAWFORD, N. M. CASPERD AND A. J. SINCLAIR Department of Biochemistry, Nuffield Institute of Comparative Medicine, Zoological Society of London, Regents Park, London NWI 4RY, England
(Received 14 August 1975) Abstract--l. The parent vegetable polyenoic acids and their long-chain animal metabolites have been studied in liver and brain phospholipids in a herbivorous mammal (Kob) and a carnivore (Hyaena) of similar body size, but contrasting developments of the nervous system. 2. The fatty acid patterns of the phospholipids were found to be tissue and species specific. 3. The major metabolite of linolenic acid was docosapentaenoate (C22:5,n-3) in the Kob liver, but docosahexaenoate (C22:6,n-3)in the Hyaena. Brain grey matter of both species contained the hexaenoate. 4. Comparative studies from 30 different species demonstrated that the brain ethanolamine phosphoglycerides (EPG) had a relatively constant fatty acid composition despite wide variations in the fatty acid composition of liver EPG.
INTRODUCTION
The phosphoglycerides from brain grey matter of m a m m a l s are rich in long-chain metabolites of linoleic a n d linolenic acids (man: O'Brien et al., 1964; rat: Kishimoto et al., 1969; whales a n d dolphin: Lesch, 1969; guinea pig: Crawford & Sinclair, 1972; squirrel monkey: Sun & Sun, 1972). Linoleic (C 18: 2,n-6) a n d linolenic (C 18:3,n-3) acids c a n n o t be made by animals (Holman, 1968) b u t are found in a b u n d a n c e in plant leaves a n d seeds (Hitchcock & Nichols, 1971). These C18 polyenoic fatty acids are partially chain-elongated a n d desaturated by animals to C20 a n d C22 fatty acids with 4, 5 and 6 double b o n d s (Mead, 1968; Klenk, 1972). Hence, the distribution of polyenoic fatty acid types in plant and animal cells is very different and there is a correspondingly wide variation in the dietary polyenoic fatty acids of different species (Crawford & Sinclair, 1972). We wish to report a remarkable constancy of the fatty acids of the brain grey matter ethanolamine phosphoglycerides despite a wide variation in the corresponding fractions from liver. METHODS
Animal species
Lipid extraction Samples of liver, semi-tendinosus muscle and a sample of the brain were stored at -20°C until analysed. The grey matter was separated from white matter in the region of the motor cortex. Weighed samples of frozen tissues were homogenised (M.S.E. Top drive) under nitrogen in twice the volume of cold (+4°C) chloroform-methanol (2:1) containing 10 mg/1 of recrystallised 2:6 di-t-butyl-pcresol as an anti-oxidant. The tissue samples were extracted twice and the combined extracts washed with 0.9% saline and evaporated to dryness at reduced pressure.
Phospholipid separation Aliquots of the lipid extracts were separated into the phospholipid fractions by thin-layer chromatography (TLC) on silica gel G using chloroform-methanol-7 N ammonia (65:30:4.5) as the solvent system. The fractions were located by dichlorofluoroscein (0.2% in methanol) and the use of adjacent standards on the same plate. The EPG and choline phosphoglycerides (CPG) bands were scraped from the thin-layer plates and transmethylated with 5% H2SO 4 in methanol at 70°C for 3 hr. The EPG and CPG fractions of muscle and brain are mixtures of diacyl-phosphoglycerides and plasmalogens (alkenyl, acylphosphoglycerides); acidic methanolysis of such a mixture produces dimethyl acetals (DMA) from the fatty aldehydes (Sun & Horrocks, 1968) and fatty acid methyl esters from the fatty acids. The DMA were included in the analysis.
Fatty acid identification The Uganda Kob (Adenata Kob) and the spotted Hyaena (Crocuta crocuta) were chosen for this study on The methyl esters and DMA were separated using a Pye the grounds of availability and similarity in adult body Gas Chromatogram (Series 104, W. G. Pye & Co. Ltd.) size. Six male adults and one female of each species and an $6 Research Chromatograph (Fisons Ltd.), both between 4 and 6 years old were selected from the Ishasha incorporating flame ionisation detectors. In both instruReserve on the borders of the Queen Elizabeth National ments, 5 ft glass columns were used with the packings of Park and from the Tonia/Kaiso flats, Uganda. The animals 10% polyethylene glycol adipate (PEGA) or 10~, Apiezon were shot with a rifle and tissues were frozen using solid M, both on 100-200 mesh celite and run routinely at CO2 within 4 min after killing. Body weights (without sto184.5 and 210°C, respectively, with an argon flow rate of mach contents) were 73 ___5.2kg for the Kob and 52 ml/min. A copolymer of ethylene glycol succinate with 64 + 4.8 kg for the Hyaena (mean + S.E.). Tissues of other methyl silicone at 10% on 100-120 mesh gas chrom P wild mammals were obtained from several sources (Table (EGSS-X) was also used under the same conditions at a 5). temperature of 190°C. The relative retention volumes of 395
396
M.A. CRAWFORD,N. M. CASPERDAND A. J. SINCLAIR
unsaturates to saturates is larger on EGSS-X by comparison with PEGA. This difference in performance characteristics enabled overlap ambiguities such as the coincidence of nervonic acid (C24:1) and clupanodonic acid (C22:5,n-3) on PEGA to be resolved; conversely on EGSS-X the eicosaenoic acid can coincide with linolenic acid but separates with a longer retention time on PEGA. The Apiezon column was used to establish carbon chain length of the methyl esters. Argentation chromatography was employed to confirm identity by separation of the methyl esters based on the degree of unsaturation, followed by hydrogenation to establish carbon chain length. The retention time on PEGA and EGSS-X for fatty acid methyl esters with the same carbon chain length increases with increasing degrees of unsaturation and is also dependent on the length of the saturated carbon chain measured from the methyl end. This characteristic enables the positioning of the double bond sequence to be determined (Ackman, 1969). A methylene interrupted sequence of double bonds was assumed when there was agreement of relative retention values with standards on both PEGA and EGSS-X, or with separation factors when standards were not available. Standards of eicosapentaenoic (C20:5,n-3) and arachidonic acid (C20:4,n-6) were obtained from the Hormel Institute. We were given the C22:4,n-6 by Professor D. A. van Dorp (Vlaardingen Research Laboratories, Unilever, Holland), the purity of which had been determined to be better than 98~o. The identity of the C22:5m-3 was established by the use of separation factors and by combined gas liquid chromatography--mass spectroscopy, and C22:4,n-6 by argentation chromatography and confirmed by mass-spectroscopy through the assistance of Associated Electrical Engineers. Docosahexaenoic acid (C22:6,n-3 with double bonds in the 4, 7, 10, 13, 16 and 19 positions) is readily available from cod liver oil which was kindly supplied by British Cod Liver Oils (Hull & Grimsby) Ltd.
Table 1. Phospholipid composition of muscle, liver and brain grey matter from the Kob and Hyaena
Species
ayae~ ~ob
Hyae~ Kob
Tissue
The nomenclature used here to describe the fatty acids follows the convention of Ca:d,n-x, where a refers to the carbon chain length, x the length of the saturated carbon chain from the methyl end and d the number of double bonds. Because of the increasing need to refer to the docosahexaenoic acid with methylene interrupted bonds in positions 4, 7, 10. 13, 16 and 19, it was felt worth introducing a trivial name for this acid and we suggest "Cervonic acid". Similarly, there is a frequent need to refer to the short chain polyenoic acids, linoleie and linolenic acids, as a specific group and separately to their chain elongation and desaturation products with 20 or 22 carbon chain lengths and two to six double bonds. Hence we shall refer to the former group as the short chain polyenoates (SCP) and the latter group as the long chain polyenoates (LCP). The results are expressed as g/100g (i.e. wt ~o) of the total fatty acid and aldehyde within the individual phospholipid fraction. In most instances data have been collected from more than one representative of each species, but in the interest of brevity the data corresponding to one of each species is recorded here except for those on the Kob and Hyaena. In all cases there was close agreement within an individual species suggesting a degree of species specificity. Whilst it was possible to obtain the tissue samples within 0.5-4min after death this was not so with the human samples which were obtained at post-mortem.
Molar Percentage
EPG
PC
Brain
26.1
35
34
14
13
Brain
25.3
55
36
ii
16
Liver
16
29
45
5.2
9.4
Liver
12
27
51
7.6
8.I
PS
SPH
there were quite marked species variations in the acyl group of liver and muscle phospholipids. The brain phospholipids were remarkably constant (Tables 2-4). The K o b liver had more C18:2,n-6 and C18:3,n-3 in the phospholipids compared with the Hyaena but arachidonic and the C22 polyenoates were present in higher proportions in the Hyaena liver than in the K o b (Table 2). In the K o b liver phospholipids, the major long chain derivative of linolenic acid was clupanodonic acid (C22:5,n-3), but cervonic acid (C22:6,n-3) in the Hyaena. This difference is also observed for the muscle phospholipids from these two species (Table 3). The analysis of one liver from a female of each species has shown a distribution of Table 2. The proportions of linoleic, linolenic acids and their long-chain derivatives in the liver phospholipids from the Kob and Hyaena
Fatty Acids
T er minoloy y
Total phospholipid g/lO0 g dry weight
Liver EPG Kob
10:z,=-6 20:2,n-6 20:5,~-6 20:4,n-6 22:4,n-6
~6,5,~-3 20,5,~-3 22:5,n-3 22:6,~-5
Liver CFG
Hyaena
Kob
Hyaena
8.2+0.5 0.5+0.06 1 .i+o .o6 12 +-0.4 I.C+0.1
5.0+0.1 0.6_+0.07 1.o*_o.o5 15 +0.5 O.}+0.o1
9.1+0.8 0.2_+0.01 o.6+O.Ol 6.0+_0.07 1.0+0,06
4.3+0.2 0.6+0.01 5.1+0.06 12 +0.4 0.6+o.01
4.4_+0.5 3.8+_o.i ii +o.i i. 6+-o.03
2.~0.09 0.4+-O.l 4.2+0.1 14 -+0.6
4.9*_0.5 4.8_+0.5 8.6z_o°i 1.1+o.2
2.7+_o.o8 o.smO°Ol 3.0+0.05 7,6+0.2
Results expressed as g fatty acid/100 g total fatty acids. Mean + S.E.M. for 6 samples of liver from each species. Table 3. The proportions of linoleic, linolenic acids and their long-chain derivatives in the muscle phospholipids from the Kob and Hyaena Fatty Acids
}@~scle E ~ Kob
10:2,~-6 2o: 5,n-6
20,4,~-6 18:3,n-3 20:5,n- 3 22:5,n-5 22:6,n-5
]~scle CPG Hyaena
Kob
Hyaena
II +--0.6 0.3+-0.05 10 +-0.8
3.1+-0.6 1.6+-0.1 15 +0°4
13+_1.6 0.4_+0.03 7.6+-0.1
4.3+-0.7 1.1+0.07 9.2+_0.1
3.2±0°4 2.1~0.0~ 7.2+-0.4 0.8-+0o01
O.9±o.05 1.0~0.04 5~0-+0.6 6.1-+0.3
3.9±0°7 1.6+-0.08 5.6+0.O9 0.4-+u°02
1.8+-0.1 i.i+-0.05 4.8-+0.2 4~7+-0.7
RESULTS
The content of total phospholipid and of the major phospholipid fractions in liver and brain from the K o b and Hyaena were similar (Table 1). However,
Results are expressed as g fatty acids/100g total fatty acids and aldehydes (see Methods). Mean _+ S.E.M. for 6 samples of muscle from each species.
Long chain metabolites in herbivores and carnivores Table 4. The proportions of linoleic, linolenic acids and their long-chain derivatives in the brain grey matter phospholipids from the Kob and Hyaena Fatty
Table 5. Sources of the material used in this study Groups
Source
CPG
EPG
Acids Kob
Hyamm
Kob
I~erm
18:2,n-6 20:2,n-6 20:5,n-6 20:4,n-6 22~4,n-6 22:5wn-6
0.6 0.~ 0.6 12 8.0 0.5
0.I 0.2 0.3 16 5.9 i.O
0.6 0.~ 0.5 8.6 1.5 0.9
0.4 0.2 5.8 2.4 1.0
18:5,n-3 20:5pn-5 22:5,n-3 22:6,n-5
0.4 1.5 1.9 25
0.2 0.9 1.6 24
0.2 I.i 3.2 5.2
0.2 0.7 1.8 7.5
_
397
Results expressed as g fatty acids/100 g fatty acids and aldehydes (see Methods). One brain sample from each species analysed in triplicate and the results are presented as the mean.
the fatty acids similar to that seen in the male animals. The brain grey matter phsopholipids in both species (Table 4) were characterised by the presence of long-chain polyenoic acids (C20 and C22), but only trace amounts of the parent C18 polyenoic acids. The EPG fraction from grey matter of both species was of particular interest as the principal fatty acid was cervonic acid, which was not the case in the Kob liver lipids. In the liver, muscle and brain from both species, the EPG fraction consistently contained a higher proportion of the long chain (C20 and C22) polyenoic fatty acids by comparison with the CPG fraction (Tables 24). As the EPG fraction of liver and brain grey matter was the richest in the long-chain polyenoic acids we selected this fraction for study in a variety of different mammalian species. The location from which these species were chosen is given in Table 5. From Tables 6 & 7 it can be seen that there were marked differences in the liver EPG but the brain EPG values were similar. In the brain, EPG was rich in LCP in the grey matter, but not in the white matter (M.A.C. unpublished data). As far as possible the samples of brain were taken from the motor cortex and were principally composed of grey matter. However, in small mammals this was not always possible and the data representing EPG from whole brain are indicated in Table 6; the consequent mixture of EPG would tend to dilute the LCP component. It might have been possible to overcome this difficulty by separating brain cells, but in this first analysis it was decided to measure whole tissue in view of the instability of the LCP. In the brain EPG the major polyenoic constituents were the LCP which appeared in the proportions of C22:6 > C20:4 > C22:4 > C22:5,n-6 and n-3 > C20:5. The percentage of C22:6,n-3, C20:4,n-6 and C22:4,n-6 averaged 21.4 + 0.82, 11.5 + 0.46 and 5.72 _+ 0.3 in data for the 28 species in Table 6 (mean Yo +- S.E.M.). By contrast, the parent C18 fatty acids of vegetable origin, linoleic acid (C18:2,n-6) and lino-
Small I,~ma I s Rat (Rattus sp.) Guinea-pig (Carla porcellus) Hamster (Cricetus cricetus) Steppe Len~ning (Laguras) Viscacha (Lagostomus maximus) Cuis (Galea musteloides) Casaragua (Proechimys guairae) Rock Hyrax (Procavia capensis) Fruit-eating Bat (Megachiropterus) Large Non-ruminants Wart-hog (Phacochoerus aethiopicus) Elephant (Loxodonta africana) Horse (Equus caballus) Zebra (Equus burchelli) Large Ruminants Topi (Damsliscus korrigum) Bu/falo, woodland (Syaoerus caffer) Hartebeest (Alcelaphus buselaphus) Elana (~uro~-agu~
oryx)
Giraffe (Giraffa camelopardalis) Red ~eer (Cervus el~phus) Ox (Bos taurus)
Laboratory, N.I.C.M.
Kilimanjaro, Tanzania Makerere, Uganda Ishasha, Uganda Newmarket, U.K. South Karamoja, Uganda, and Z.S.L. Ishasha, Uganda South Karamoja, Uganda ,, ,, Z.S.L. London, U.K.
Carnivores Cat (Fells catus) Civet (Viverra civette) Leopard (Panthera pardus) Lion (Panthera leo) Canadian Timber Wolf (Canis lupus)
Laboratory, N.I.C.M. Bulemezi, Uganda Ishasha, Uganda Z.S.L.
Primates Marmoset (Leontideus rosalia) Patas Monkey (E~ythrocebus pates) Vervet Monkey (Cercopithecus aethiops) mamma (~omo s s p i ~ )
Laboratory, N.I.C.M. N.I.C.M, and Madl, N. Uganda (origin, Uganda) London
Marine Mammals Bottlenosed Dolphin (Tursiops truncatus)
Z.S.L.
lenic acid (C18:3,n-3) had averages of 0.93 + 0.1 and 0.26 + 0.04 respectively. That is, the brain was characterised by a high LCP and low SCP content. This degree of uniformity of brain EPG fatty acid profile was not found in the liver EPG. For example, the mean linoleic and linolenic acid proportion in the liver EPG of small mammals was 13.1 + 1.8~o and 1.3 + 0.54~o which is many times greater than that which was found in the brain. The arachidonic acid of 10.8 + 1.5~o and the cervonic acid of 19.5 + 2.5~o were similar to the levels found in the brain but the docosatetraenoic acid (C22:4,n-6) was much lower in the liver EPG at 0.5~o ___0.12~o. The guinea-pig reaches an adult body weight of about twice that of the rat. In the case of the rats and guinea-pigs studied here, both were fed the same diet, yet the EPG of the guinea-pig contained significantly less LCP but more SCP than was found in the rat liver EPG. The contrast between brain and liver EPG was even greater in the case of the large non-ruminants or what might loosely be referred to as herbivores, except that the warthog is in principle omnivorous even if, in our experience, it is predominantly a herbivore in practice. In this group the linoleic acid proportion was remarkably high. Although this group only contains four species the analyses represent a total of 15 animals and there was close agreement in the data obtained within a given species. The liver EPG linoleate of the five warthog averaged 23.2 + 1.6~o and the four elephant 11.0 + 1.8~o. The mean linoleate of all four non-ruminant species was 24°/,. The zebra was of special interest because of the
398
M.A. CRAWFORD,N. M. CASPERDAND A. J. SINCLAIR Table 6. Brain grey matter EPG polyenoic fatty acids n-6
T
12 12 l0 II
6.5 7.1 4.9 7.2
0.8 0.6 0.9 0.4 0.2 0.7
II I0 ll 12 14 14
0.6 0.7 0.8
0.2 o.I
i,2 1.5
0.5 0.2
0.7 O°6
2.1
0.5
0.6
0.6 0.9 0. 9 1°5
25 19 18 25
7.2 6.9 6.3 6. 5 7.0 6.2
o.8 1.2 0.8 I.i 1.2 o.9
o.7 0.7 0.5 I,I 0.8 0.4
0.6 0.8 0.6 0,5 0.4 o.7
Oo7 0.2 1.2 0.4 0,5 0.5
17 25 22 16 21 24
13 14 14
6.7 5.9 7.1
I.i 1.5 1.o
0.} O.4
0.8 0.8 0.5
0.8 0.5 0.7
27 26 22
0.5
9
5.0
0.5
o,4
16
0.I O.1 0.5 1.0
ll 6.8 14 14
4.7 5.3 6.8 8.O
1.6 1.2 0.5 2.2
0.i 0.8 0.9
0.5 0.6 0.5 1.6
29 19 21 25
0.i
6.9
5.6
0.2
0.8
2.5
27
Lar6e Non-Ruminants Wart-hog 1 Horse 4 Zebra 2 Elephant 3
0.9 1.0 O.1 i.I
0°l 0.4 0.1 0.2
0°2 o.7 0.5 0.8
i 1 6 1 5 1
1.5 0.8 1.2
0.5 0.3 0.6
2.5
0.5
1.7 1.4
0.2 0.2
2 1 1
O°9 1.2 ~ 0.7
0.2 0.3
Primates Marmoset Vervet
Patas Man Marine b~m~ Is Dolphin
I 1 1 4 5
0.8 0.i 0.6 O.5
2
0.7
0.1 0.6 0.2 O.7
i
22:5 0.5 0.6 0.2 0.7 i.i 1.5 0.6 1.4
0.7 0.i 0.3 0.5
Carnivores Cat Civet Leopard Canadian Timber Wolf
-
20:5 0.2 0.6 0.4 0.4 0.4 0.5 0.9 o°5
20:5 0.5 i.i 0.5 1.O 0.9 O. 5 0.4 0.6
Ox Buffalo Eland Giraffe
n-5
18:3
20:2 0.i 0.2
Red Deer
i
22:5 0.6 2.9 0.2 1.5 0.7 1.2 0.6 2.5 0.5
18:2 0.5 1.6 1.5 1.5 1.4 0.5 0.6 0.9 0.5
Larl~e Ruminants Hartebeest
,
22:4 5.1 7.9 4.2 4.5 6,1 4.7 2.9 5.2 1.1
Small Mammals n Rat i0 Guinea Pig 10 Hamster 2 Steppe Le~mning 6 Viscacha l Cuis i Casaragua i Fruit-eating bat 2 Vole 1
20:4 II 17 9.6 i0 12 8.5 9 •7 15 ii
0.5
0.i
22:6 21" 19 22 29* 16 13" 15" 21 2O*
A similar difference between liver and brain EPG was seen in the large ruminants in which the liver fatty acids were characterised by a high SCP and low LCP content. In the eight species studied the mean linoleate and linolenate proportions were 10.2 ___ 1.5% and 2.8% respectively. Liver arachidonic acid levels
remarkably high linoleate (47 and 45%) in both specimens studied. Another striking difference between the liver and brain EPG in this group was the very low proportion of cervonic acid (1.5%) which characteristically constituted about one-fifth to one-quarter of the fatty acids of cortical grey matter EPG.
Table 7. Liver EPG polyenoic fatty acids r
o.1 0.2 0.I 1.3
o.6 1.8 0.3 4.5
12 6,2 4.2 i0
o.5 o.5 o.1 0.4
0.i 0.4 0.I 0.i
5.5 0.9 1.6 2.8
5.7 1.0 0.5 5.8
5.o 2.8 1.0 ll
1.6 5.6 0,2 0.6
Red deer
5 4 2 8 4 4 4 2
14 ii 5.1 4.0 9.7 8.5 12 17
0.2 0.2 0.5 0.9 0.2 0.2 0°2 0.9
0.3 0.4 0.5 1,2 0.2 0.5 0. 5 0.9
9.2 14 11 16 13 14 12 16
0.5 0.7 0oi 2. 5 0.5 0.6 i,i 1.9
0.5 0.5 0.I 0.6 o.l 0.2 0.8 0.8
4.0 5.3 3.5 1.0 3.6 2.8 2.1 1.9
4.1 2.9 7.8 5.1 2.6 1.9 2.1 2.7
9.0 ll 8.5 II 12 7.8 9.~ 8.1
0.8 0.9 0.5 2,5 1.8 6.4 0.8 4°8
Ox (domestic) Buffalo Eland Giraffe Camel Carnivores ~ t i c ) Civet Leopard Li on
2 3 2 I
5.5 5.4 5.8 2.9
0.2 o.5 0.I 0.5
1.4 0.5 O.9 1.7
18 15 15 16
1.5 5.6 2.4 5.6
1.0 1.1 0.7 1.4
1.5 2.0 1.6 0.2
0.9 2.0 O.4 1.5
0.5 0.8 2.O 7.7
22 20 18 9.4
Pri~tes Marmoset Vervet
Patas Human (adult) Foetus
I 2 4 3 i
i0 9.2 9.0 6.3 Ooi
o. 5 1.3 0.5 0.8 o.75
o,6 o.8 3.1 4.2 1.54
23 17 18 16 5o
0. 7 O.5 0.8 5.5 5.4
i,3 o.4 o.5 1.5 1.4
I,i 0.5 2.3 0.2 0.5
o.4 5.2 2.5 1.6 0.5
o.5 2.6 4.8 4.o 0.5
8.2 5.9 8.9 7.7 15
b~rine ~rm~al Dolphin
2
1.7
o.2
13
o.9
o.9
O.1
8.2
1.5
ll
-
20:4 21 11 13 7.6 ll 14 4.1 9.5 ii 5.5
22:4 0.6 1.3
22:5 0.4 0.9
0.5
0.5 0.5 0.2 0.4 0.2
n-3
22 17 47 lO
11
20:2 o.i O.1
,
o.I 0.3 0.5 0.2 o.i 0.2
Large Non-Ruminants Wart-hog 5 Horse(domestic) 4 Zebra 2 Elephant 4
18:2 6.0 25
,
16 20 16 8.5 16 8 7.2
n I0 i0 Hamster 2 Steppe Lemming 6 Viscacha i Rock ~ r a x i Cuis i Casaragtm i Fruit-eating bat 2 Vole i
Large Ru~nants Topl Hartebeest
n-6
20:3 0.5 0.5 0.5 0.5 0.7 1.O 0.2 0.l I.i 0.2
Small M~mmals Rat Guinea Pig
18:3 o.l 5.4
20:5 1.5 2.2
22:5 5.5 2.8
0.5
0.3
o.7
1.2
0.9 0.3 0.2 0.2 0.05
0.7 0.6 3.2 0.3 0.2 0.6 1.8
1.7 1.6 0.6 1.7 1.9 1.4 1.0
i.o 1.1 0.9 2.2 0.5 5.4 2.2
22:6 21 10 23 21
22 21
52 15 5,9 26
Long chain metabolites in herbivores and carnivores
399
were similar to the brain at 13.1 + 0.84~o but the cervonic acid proportion was only 2.3 + 0.78~o. It was of interest that in both the large herbivore groups the clupanodonic acid (C22:5,n-3) was greater than the cervonic acid (C22:6,n-3). In the large ruminants the clupanodonic acid averaged 9.6 + 0.56~o. The carnivore liver EPG was characterised by a significantly lower proportion of SCP and a significantly higher proportion of LCP in comparison with the herbivores. This difference in profile is the same as was found for the direct comparison between the Kob and Hyaena. In summary, this comparison of the EPG of liver from large herbivorous mammals established them to be relatively rich in SCP whilst the carnivore livers were relatively rich in LCP but not SCP. The profile found in the primates included both SCP and LCP. Despite this diversity of liver EPG the brain EPG was similar in all species. The opportunity arose to study two dolphins. Whilst the land food chain provides both the n-6 and n-3 series of acids the marine food chain is predominantly concerned with n-3 fatty acids (Ackman, 1964). Because of this extreme difference between the food chemistry on land and in the sea it was felt that the dolphin brain might be different. It was therefore surprising to find that not only was the brain EPG similar to that of land mammals, but also the liver EPG of this species accumulated both series of acids, suggesting either a specialised food selection pattern or an ability to concentrate the n-6 series of acids from the small amounts available in the principal fish species. In Table 8 the total lipid content of liver and brain cortex grey matter is expressed on a dry weight basis. The high proportion of the lipid in the brain illustrates that lipid accounts for the highest proportion of brain solids.
interaction of lipid or polyhydric alcohols and trace minerals or complex prosthetic groups with the protein molecule. However, it is generally believed that the functional unit is primarily dependent on the initial code for the amino-acids and the unique differences between the amino-acids, although the chemical differences between many of the individual aminoacids is not great. For example, the essential aminoacid leucine is distinguished from the non-essential amino-acid alanine only by two methyl groups; leucine and isoleucine have identical empirical formulas yet are both referred to as separate and non-interconvertible essential amino-acids. Similarly, there are both essential and non-essential fatty acids in the structural lipids and the distinction between the chemical structures of the fatty acids does not appear to be great, at least on paper. Oleic (C 18 : 1,n-9) is distinguished from linoleate (C 18 :2,n-6) by one double bond and linolenate by an additional double bond (C18:3,n-3); however, the positioning of these and the number of double bonds in the fatty acid not only affect their physical properties (e.g. melting point, Chapman, 1972) but also their biological properties, for animal systems are unable to insert double bonds in the n-6 and n-3 positions. This means that animals can synthesise oleic but not linoleic nor linolenic acids and cannot convert linoleic to linolenic acid (Mead, 1968). Linoleic and linolenic acids occur in plants and there is evidence that they both exhibit "essential fatty acid" properties (Houtsmuller, 1973), Furthermore, the LCP have different activities to the SCP. For example, arachidonic and cervonic acids are incorporated some 10~0-times faster into the developing brain by comparison with linoleic and linolenic acids (Sinclair & Crawford, 1972; Sinclair, 1975). Both linoleic and linolenic acids are metabolised in the liver to derivatives in which the chain has been elongated at the carboxyl end of the molecule. The DISCUSSION liver enzyme systems are also capable of introducing additional double bonds between the carboxyl and The two principal building classes of biochemicals the existing methylene interrupted sequence. As a in the cells of soft tissues in advanced forms of life result the liver makes available two families of highly are the proteins and lipids. Protein is quantitatively unsaturated fatty acids, i.e.: the most important in muscle whilst the lipids are quantitatively the most important in the brain and 18:2,n-6 ~ 18:3,n-6 ~ 20:3,n-6 --~ 20:4,n-6 nervous system. Proteins are mainly built from 20 22:4,n-6 ~ 22:5,n-6 amino-acids, eight of which are usually considered 18:3,n-3 ~ 18:4,n-3 --~ 20:4,n-3 --~ 20:5.n-3 essential to animals, that is, they cannot be synthe22:5,17-3 ---, 22:6,n-3 sised by animals or cannot be synthesised fast enought to meet the demands of cell growth. In the structural lipids these polyenoic fatty acids The complexity of the protein system is dependent are found in the /3 position of the phospholipid. In on the sequence of the amino-acids. Further elabo- general all the main polyenoic acids underlined are ration of the structure is achieved by the folding and found in the structural lipids of animals although interaction of the polypeptide chains and finally the within any given animal species a tissue specific patfunctional unit of the cell is dependent on a further tern seems to exist (e.g. adrenal cholesteryl esters: Table 8. Mean lipid content of brain cortex and liver per 100 g dry wt LIV~
No.
Mean
21"
22.9
BRAn~ Grey Matter
+
S.E.
No.
2.2
20
Mean 51-9
S.E. +
1.36
* Excludes small mammals where brains were not separated into white and grey.
Ostwald et al., 1964; Carney et al., 1971 and testis lipids: Bieri & Prival, 1965). Our results confirm that in the brain neither linoleic nor linolenic acids appear in significant amounts and in a wide range of advanced mammalian species the brain polyenoic fatty acids were restricted to the LCP of C20 and C22 carbon chain length: regardless of species, food selection pattern or liver tissue lipids, the pattern of polyenoic acids found in the brain was substantially the same. This fact has some interesting implications;
400
M.A. CRAWFORD,N. M. CASPERDAND A. J. SINCLAIR
the LCP are not commonly found in plant lipids and therelore the production of a given structural lipid profile of fatty acids is dependent both on the metabolic sequence and on exogenous supply. Both the manner of supply and metabolism demonstrate that the principles of assembly of lipids and proteins during cell growth are different in a fundamental respect. In protein assembly, individual amino acids are incorporated in the sequence. In lipid assembly the parent fatty acids have to be metabolised further and both parents and metabolic products are incorporated' into the cell structural lipids. That is, the difference amounts to the use of discrete individuals (amino acids) as opposed to the use of families (fatty acids). A further difference lies in the exogenous source of lipids and amino-acids. Photo and microbial synthesis produces all the amino-acids found in animal proteins; plants provide the parent SCP. This means that there are two exogenous sources for members of the essential fatty acid families found in animal cell structures: plants provide SCP and animal products LCP. If the metabolism of SCP to L C P was rapid in animals, the likelihood of animals accumulating significant amounts of SCP in their tissues would be low. In all animal species we studied, significant amounts of SCP did accumulate in the E P G ; in the cell structural lipids. Indeed, it has been argued that a diversity of fatty acids is an important structural feature of these lipids (Crawford & Sinclair, 1972) and this diversity would not be possible if instant conversion of SCP to L C P did occur. Indeed, from the evidence of the liver lipids presented here, it is plausible that the small, but not necessarily the large animals have the ability to elaborate a complete fatty acid profile. (cf. large ruminants and non-ruminants with small mammals Table 7). It was especially interesting that it was the fastest growing animals and those which reach the greatest adult body weights in which the chain elongation and desaturation process did not appear to reach completion in the liver and muscle tissue. Yet even in these species only the fully desaturated L C P were found in the brain. The fast growing large mammals have the smallest brains in proportion to body weight (Spector, 1956). Both these facts are consistent with the suggestion that the factors concerned with brain growth need not be the same as the factors concerned with body growth; the plausibility of this suggestion is supported by Sacher's (1968) foetal growth analysis, which illustrates wide contrasts in body and brain growth in different species. Also the buffalo, zebra and horse can achieve body weights of about 500-1000 Kg in 3-4 years and have a relative brain to body weight of 0.06%, whereas man only achieves a body wt of 7 0 K g after 18 years of growth and has a brain body wt ratio of 2%. As the brain is predominantly lipid (Table 8) this suggestion might be expressed in biochemical terms by saying that protein synthesis could proceed at a different rate to lipid synthesis. The accumulation of both SCP and LCP in liver structural lipids, the failure of large, fast-growing herbivores to fully desaturate liver and muscle LCP
despite an obvious abundance of the SCP precursors and the progression of the polyenoic acid profile from herbivore to carnivore towards the LCP could be explained by a slow rate of conversion of SCP to LCP, rate-limited by the desaturation step (Hassam et al., 1975). The remarkable specificity of the brain polyenoic acids raises a number of interesting questions pertinent to the evolution of large animals and the obvious disparity between the accumulation of body and brain weight. The biochemical evidence presented here suggests that the long-chain 15olyenoic acids could be a qualitative determinant of brain development in higher animals. Acknowledgements We are grateful to Mrs. Lynne L. Springett, Miss P. Stevens and Mr. G. Williams for skilled technical assistance. We are also pleased to record our appreciation of the assistance given by the Game Departments of Uganda and Tanzania without whom this work would not have been possible. We are indebted to Professors J. Boldingh and D. A. van Dorp of Unilever for technical discussions and for fatty acid standards, and to Unilever Vlaardingen for financial assistance. We are also grateful to Dr. I. Keymer and Mr. D. Jones of the Zoological Society of London for assistance with specimens.
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