J . Sci. Food Agric. 1982, 33, 421430

The Influence of Dietary Branched-chain Fatty Acids on the Fatty Acid Composition of Tissue Lipids of Rats

Alistair Smith, A. Kenneth Lough and Charles R. A. Earl

Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB

(Manuscrbt received 22 April 1981)

Branched-chain fatty acids (BCFA) were prepared as a concentrate from the adipose- tissue lipids of barley-fed lambs. The BCFA was included in a stock Oxoid ration at levels of 2.5-15% by weight and these diets were given to appetite to female weanling rats for up to 14 days. The influence of the diets on inter alia growth-rate and fatty acid composition of tissue lipids was compared with the effect of the same stock diet with inclusions at a level of 5% by weight of 3-methylhexadecanoic acid (3-MHD), 4-methylhexadecanoic acid (CMHD), 3,7,11,15-tetramethylhexadecanoic (phytanic) acid and n-hexadecanoic (palmitic) acid which, with the stock ration, served as control. Rats receiving diets containing up to 7.5 % BCFA grew almost as well as did those given palmitic acid whereas the animals offered the diets containing 3-MHD, 4-MHD or 10% BCFA barely subsisted. Rats given 10% BCFA or phytanic acid became moribund within 3 days. BCFA accumulated to widely varying degrees in the lipids of the main internal organs. The higher proportions of these acids constituted some 40-60% of the total fatty acids of the lipids of kidney, liver and heart and were associated with diets containing 10% and 15% BCFA and 5 % 3-MHD. The lower proportions of the branched acids in the lipids of these organs were in the range 15-25 % of the total fatty acids and related to diets that included 2.5% BCFA and 5% 4-MHD. In contrast to the changes in fatty acid composition of the lipids of the main internal organs, those of the central and peripheral nervous systems were only minor, the proportions of branched acids amounting to some 3 % in brain tissue and up to 14% in sciatic nerve. Clearance of branched acids from tissue lipids of the main internal organs was almost complete after the animals were returned to the basal diet for 14 days but the loss of branched acids from nerve tissue was evidently less rapid.

1. Introduction

Investigations relating to the effect of feeding high-energy rations to ruminants revealed that the subcutaneous adipose tissues of lambs given diets containing about 90% rolled barley were ab- normally soft.' The soft-fat condition was found to be associated with the presence of branched- chain fatty acids (BCFA) in proportions of up to 10% of the total fatty acids of the adipose tissue triacylglycerols.2 Detailed analysis using high resolution gas chromatography linked to mass spectrometry showed that the BCFA were of chain lengths C~O-CIS (odd- and even-chain) and consisted for the most part of monomethyl-substituted fatty acids together with minor proportions of di-, tri- and tetramethyl-substituted fatty acids;3r4 ethyl-substituted fatty acids were also identified.5 Some 180 components have been identified and in all but two minor constituents, substitution occurred on the even-numbered carbon atoms (relative to the carboxyl group). Monomethyl- substituted fatty acids also occur, albeit in very small proportions, in the tissue lipids of grass-fed sheep and in butterfat.6

The presence of methyl-substituted fatty acids in relative abundance in animal tissues which serve as food for man raised the question as to the fate of these acids on assimilation into the

0022-5142/82/05O0-0421 $02.00 0 1982 Society of Chemical Industry

421

422 A. Smith et al.

animal body. Thus, BCFA in the form of a concentrate were prepared from the triacylglycerols of subcutaneous adipose tissue of barley-fed lambs and were included in a stock diet at a level of 5 % to weanling female rats. The influence of the BCFA was compared with the effect of inclusions in the same stock diet, of 3,7,11,15-tetramethylhexadecanoic (phytanic) acid, 3-methylhexadecanoic acid (3-MHD) and 4-methylhexadecanoic acid (4-MHD); the stock diet with and without the inclu- sion of n-hexadecanoic (palmitic) acid served as controls. The choice of 4-MHD as a model compound for comparison with BCFA was based on the fact that among the plethora of BCFA forming the concentrate the 4-methyl configuration was by far the most common as was the chain length of 16 carbon atoms.4 Phytanic acid was selected because it is one of the few methyl-substituted hexade- canoic acids that has been studied in some detail in a nutritional context. Lastly, the inclusion of 3-MHD in the investigation afforded a link between 4-MHD and phytanic acid in that it is a positional isomer of the former and an analogue of the latter.

Observations were made on food intake and on growth rate of the experimental animals and on the fatty acid composition of the lipids of brain, peripheral nerve and the main internal organs. An assessment was made of the clearance of the experimental acids from the tissues when the animals were returned to the basal diet. A preliminary account of this work has been published previou~ly.~

In anticipation of a more definitive feeding trial involving female rats and their progeny, BCFA at levels of 2.5-15 % by weight of the diet were also given to weanling rats.

2. Experimental 2.1. Synthesis of branched-chain fatty acids 3-Methylhexadecanoic acid was prepared by the Kolbe electrolysis of methyl hydrogen 3-methyl- glutaric acid8 and n-tridecanoic acid (Sigma Chemical Co. Ltd, London) in a cell fitted with a water jacket. The electrolysis was carried out between a mercury cathode and a rotating platinum anode9 at a current density of 2 A cm-2; methanol was used as solvent. The products of electrolysis were extracted and separated by fractional distillation in vucuo to yield an ester fraction containing 96 % methyl 3-methylhexadecanoate and 4 % methyl tridecanoate. The esters were hydrolysed and the resulting acids were treated with ureaLo to give 3-MHD in pure form; the overall yield of 3-MHD was 27 %.

4-Methylhexadecanoic acid was synthesised by two methods. In one synthetic pathway, starting with 1-bromododecane and ethyl levulinate, the desired acid was obtained via the intermediate, ~-dodecyl-~-methylbutyrolactone~~ (81 % yield). The other pathway involved the formation of 3-methylpentadecanoic acid by cross-coupling methyl hydrogen 3-methylglutaric acid and n- dodecanoic acid, followed by chain extension of the product of anodic synthesis by the Arndt- Eistert methodl2 (22% yield). The products obtained from the two methods were combined and distilled in vucuo, as methyl esters, to yield a fraction containing 92 % 4-methylhexadecanoate and 8 % methyl 3-methylpentadecanoate. The esters were converted to fatty acids prior to feeding to rats.

Phytanic acid was prepared by the catalytic reduction of phytol (L. Light & Co.) using palladium and charcoal in methanol; the resulting dihydrophytol was oxidised with Cr03 and KHS04;13 the yield of phytanic acid was 52%. The products of the oxidation were distilled in vucuo, as methyl esters, to yield methyl phytanate in pure form ; hydrolysis yielded the acid.

2.2. Branched-chain fatty acid concentrate A concentrate of BCFA was prepared on a large-scale batch basis by the following method. Lambs given a diet containing 90% rolled barley were slaughtered when they reached about 40 kg in weight. The subcutaneous adipose tissue was removed from the carcasses, rendered, dried by heating at 110°C for 6 h and filtered through muslin to remove particulate matter. In a typical process, fat (10 kg) was transmethylated by reaction with sodium (60 g) in methanol (25 litres) for 3 h. Urea (28 kg) was added to the reaction product and the mix allowed to stand overnight. The supernatant was removed by filtration yielding a non-adducting fraction consisting essentially of unsaturated esters and branched-chain esters. After evaporation of the solvent. the esters were washed with water

Fatty acid composition of tissue lipids of rats 423

and collected. At this stage, the non-adducting fractions from several batch processes were pooled and the esters (12.5 kg) containing about 30 % branched-chain components were hydrogenated (Unilever, Vlaardingen, Holland).

The saturated esters were converted to the corresponding acids and fractionated with urea (urea:acid, 2.8: l ) .10 The acids in the non-adducting fraction were collected by filtration and con- verted to methyl esters by reaction with 20 g kg-1 sulphuric acid in methanol. The esters were purified by distillation in vacuo, converted to acids and extracted into alkali to remove any residual non- saponifiable material. On acidification, a concentrate of BCFA (5 kg) containing 85 % branched- chain components in admixture with straight-chain saturated components was obtained. The structures of the BCFA in a similar concentrate from barley-fed lambs have been determined.3-5

2.3. Lipid extraction and preparation of methyl esters Tissues from within each group of animals were analysed individually except for sciatic nerve which was pooled to provide sufficient lipid for analysis. The tissue lipids of liver, heart, brain, kidney and sciatic nerve were extracted according to the method of Folch ef a1.14 Extracted lipids of the heart, brain, kidney and sciatic nerve were transesterified with 50 g litre-' methanolic hydrochloric a ~ i d . 1 ~ The transesterification products were separated into fatty acid methyl esters and dimethyl acetals by thin-layer chromatography on Kieselgel G (E. Merck AG, Darmstadt, West Germany) with xylene as the mobile phase.16 The band containing the methyl esters was scraped off the plates and the esters eluted from the adsorbent with diethyl ether. Liver lipids were separated into neutral lipid and phospholipid by silicic acid column chromatographyl7 before conversion into fatty acid methyl esters.18

2,4. Gas--liquid chromatography Methyl esters were analysed, before and after removal of unsaturated components,19 by gas-liquid chromatography (Pye Series 104 and GCD; Pye Unicam Ltd, Cambridge) using 1.5 m x 4 mm i.d. glass columns packed with either 15 % polymerised ethylene glycol adipate (Supelco Inc., Bellefont, Pennsylvania, USA) or 10% Apiezon L (Shell Chemicals Ltd, London) on Celite 545 (Shandon Scientific Co., London); the operating temperatures of the columns were 185 and 200" C, respec- tively.

3. Diets and animals

The protocol for the investigation is given in Table 1. Weanling 21-day-old females from eight litters of Hooded Lister rats of the Rowett Institute stock were randomly assigned to six groups each with five rats, and to four groups of two rats.

The experimental diets used in the study were prepared from a stock ration which comprised a milled basal (Oxoid) diet to which was added bran that was previously defatted by treatment with diethyl ether. The stock ration containing bran at a level of inclusion of 5 % was mixed with fatty acid by employing the bran as carrier. Thus, the various fatty acids and the BCFA were each coated on bran by solvent evaporation from ether solution finally to give a series of diets that contained, by weight, 5 % palmitic acid, 5 % phytanic acid, 5 % 3-MHD, 5 % 4-MHD and 5 % BCFA. These diets as well as the stock ration were offered to the six groups of five rats. The four groups of two rats were given diets that were based on the stock ration but which contained 2.5, 7.5, 10 and 15 % BCFA, respectively.

Total fatty acids constituted 2.8 % of the stock diet and 2.9 % of the basal diet. The major compo- nent fatty acids of the stock and basal diets were 18:2 (44.373, 18:l (25.1 %), 16:O (17.9%) and 18:O (6.1%); minor components included 12:0, 14:0, 15:0, 16:1, 17:0,20:4 and 22:6.

It was planned that the respective diets would be given to the experimental animals for 14 days at the end of which time the rats receiving BCFA at levels of 2.5, 7.5, 10 and 15 % would be killed as would three rats from each of the groups on the other dietary treatments. The surviving animals (one pair from each group of five) would then be offered the basal diet for a further 14 days. However, the changeover from experimental diet to basal diet had to be advanced to the eighth day for those

424

Table 1. Dietary treatments of animals on experiment

A. Smith et al.

Diet

Stock Stock; basal 5 % Palmitic acid 5 % Palmitic acid; basal 5 % Phytanic acid

5 % 3-MHD; basal

5 % 4-MHD; basal 5 % BCFA 5 % BCFA; basal 2 . 5 % BCFA 7.5% BCFA 10 % BCFA 15% BCFA

5 % 3-MHD

5 % 4-MHD

Period on diet (days) Number of

animals Experimental diet Basal diet

3 2 3

4" 3 2 3 2 2 a 2 2 2 2 2

7 -

14 14 14 14 3 8 8 8 8

14 14 14 14 14

3

One animal from each of those groups died after 2 days on the respective experimental diets and no further account was taken of them.

rats receiving 3-MHD and 4-MHD because of their poor condition by that time. The animals receiving phytanic acid and 15% BCFA became moribund on the third day and were all killed. Also, it should be noted that one of the rats given phytanic acid and one given 15% BCFA died on the second day and no further account was taken of them.

The animals were housed individually and were given the diets ad lib. with continuous access to water. The food intakes and body weights of the rats were recorded daily. Rats were anaesthetised with diethyl ether and killed by decapitation. Tissues were excised immediately, frozen in liquid nitrogen and stored at - 20°C prior to analysis.

4. Results

As already indicated marked differences were observed in the growth response of the rats to the respective diets. Growth rates of animals receiving diets containing 2.5, 5 and 7.5% BCFA were significantly lower than those of the control group given the stock diet but the range of observed values apparently overlapped that of the rate of growth of the rats given the diet which included 5 % palmitic acid (Table 2). The reduction in growth rate associated with the inclusion of BCFA may well be linked to a lowered efficiency of utilisation of the diets containing these acids as well as to reduced food intakes (Table 2).

When growth rate is considered in relation to the total quantity of each of the branched acids ingested by the rats, the adverse effects associated with phytanic acid resulted from an intake of only about 0.1 g per rat whereas the animals given diets containing 7.5 or 10% BCFA consumed about 10 g of the concentrate per rat during the 14-day feeding periods. Also from Table 2, it can be seen that the animals given 3-MHD and 4-MHD had low intakes and showed no growth.

The fatty acid compositions of the tissue lipids of rats receiving the experimental diets and latterly the basal diet for the periods specified in Table 1 are presented as appendices.a For clarity of present- ation, the composition of the major component fatty acids of each of the tissues has been selected from these data and is given in Table 4. The values given for the proportions of BCFA are composite

a The appendices which accompany this paper are deposited with the British Library, Boston Spa, Wetherby, Yorkshire LS23 7BQ and may be obtained by quoting the Supplementary Publications Number SUP 11032.

Fatty acid composition of tissue lipids of rats 425

Table 2. Food intake and weight gain o f animals given diets containing experimental fatty acids (mean values/ dietary group)

Period on diets (days)

8 days 14 days _ _ _ . . _ _ ~ ~ -. . ~ Number

of Food intake Weight gain Food intake/ Food intake Weight gain Food intake/ Diet animals (g day-1) (g day-1) weight gain (g day-I) (g day-') weight gain

. . . _ _ _ ~ ~ ~- ~

Stock 5 12. I 4 . 7 2.58 14.2 4 . 6 3.12 5 7; Palmitic acid 5 1 1 . 5 4 . 2 2.71 12.7 4 . 0 3.17 57; 3-MHD 5 6.3 0 .0 5 % 4-MHD 5 4 . 9 - 0 . 6 57; BCFA 4 9 . 7 3 .1 3.18 10.9 3 .3 3.32

7 . 5 % BCFA 2 8 . 8 3 . 0 2 .96 10.2 4 . 0 2.58 10% BCFA 2 6 . 4 1.3 4.96" 7.4" 1.9" 4 . 2 9 Standard error of n=2 f0 .59 k 0 . 2 5 k 0 . 1 3 k 0 . 4 1 k 0 . 2 4 k 0 . 1 4

n = 5 f 0 . 3 7 r t0.16 k 0 . 0 9 k 0 . 2 6 k O . 15 f 0 . 0 9

2 . 5 % BCFA 2 11.2 4 . 0 2.80 12.2 3 . 6 3.43

means (s.e.m.) n = 4 k 0 . 4 2 kO.18 k 0 . 0 9 k 0 . 2 9 k 0 . 1 7 kO.10

Overall significance of diet differences (F test) P<O.OOl P < O . O O l P < O . O I P < O . O O I P<O.OI P<O.OS --

a These: values were excluded from the analyses o f variance because the variability between the two rats on this diet was higher than the variability within the other groups. The s.e.m. are therefore not applicable to these mean values.

and represent the summation of the relative proportions of the areas under those gas chromato- graphic peaks that were attributable to methyl esters of BCFA. However, it should be noted that in the tissue lipids the proportion of branched-chain components with 14 carbons or less in the fatty acyl chain was lower than that in the BCFA in the diets.

The relative proportions of branched-chain acids contributing to the total fatty acids of the neutral lipids and phospholipids of liver and to the total fatty acids of kidney and heart increased with increasing proportions of BCFA in the diet (Figure 1). This trend apparently continued up to a level of intake of 15 % BCFA, except in the heart, despite the fact that the high proportions of branched acids in the lipids of liver and kidney of the animals receiving this diet were attained after only 3 days,

Rats which received the diet containing CMHD showed a distribution of the branched acid in the lipids of the main internal organs which was similar to that of animals given BCFA at levels of 2.5 and 5 %. In marked contrast, rats given the 3-MHD diet accumulated the branched acid in the lipids of these tissues to an extent that was comparable to that associated with diets containing 10 and 15 % BCFA. The four rats which were examined after 3 days on the diet containing phytanic acid exhibited effects that more closely resembled these observed in the animals given 3-MHD than those in the rats given either 4-MHD or BCFA (Figure 1).

In relation to the proportion of total lipid in the form of neutral lipid in liver, it can be seen from Table 3 that the animals receiving 10% BCFA, 15% BCFA, 3-MHD and phytanic acid had significantly higher proportions of neutral lipid than did the animals receiving the control diets ( P < 0.0.5).

In the lipids of brain, up to 3% of the total fatty acids were attributable to branched acids. The proportion of total fatty acids as branched acids in sciatic nerve tissue lipids was of the order 1-3 % in those animals receiving phytanic acid, 3-MHD, 4-MHD and 15 % BCFA and from 7 to 14% in animals given 2.5-10% BCFA.

The animals which were given the basal diet after the experimental diets retained only a small proportion of branched acids in the tissue lipids of the main internal organs. However, a substantial

426

70

60

50

40

u 30- 0

0

.-

2 20-

2 10 2

Lo 0-

- r O - 5 .-

0

A. Smith el al.

-

- -

-

-

14-

12

10

8 -

6 -

4 -

2 -

0-

Liver phospholipid

- -

a b c d e f g h

7 0 -

e c 6 0 - 0

u 0

2 5 0 - u

Kidney total l ip id

Liver neutral lipid

T I I

o b c d e f g h

H e a r t total lipid

a b c d e f g

Brain total lipid

Sciatic nerve total lipid

O b c d e f g h a b c d e f g h

Figure 1. The proportion of branched acids in the total fatty acids of tissue lipids o f rats given diets containing experimental fatty acids: a=phytanic acid; b=3-MHD; c=4-MHD; d=2.5”i, BCFA; e=S%BCFA; f = 7 . 5 % BCFA; g= lop/, BCFA and h = 150,; BCFA. S.e.m. are indicated by bars.

proportion of the branched acids which were originally deposited in the brain and the sciatic nerve lipids remained in these tissues when the animals were returned to the basal diet (Table 4).

The inclusion of BCFA in quantity in the fatty acid constituents of the various tissue lipids of the rats was generally accommodated by proportionate changes in the content of the normal comple- ment of tissue lipid fatty acids rather than by displacement of specific fatty acids (Table 4). However, from the ratio of octadecenoate: octadecanoate (18: 1/18:0) in the liver neutral lipids of animals receiving phytanic acid, 3-MHD, 4-MHD, 10% BCFA and 15% BCFA, there was the suggestion that A -9 desaturase activity may have been impaired. This possibility was investigated and it was shown from experiments in vivo and in vitro that the production of 18: 1 was indeed reduced in the presence of BCFA.2Q21

5. Discussion 5.1. Influence of experimental diets on growth rate The findings in the present study indicate that rats given a nutritionally adequate diet containing up to 7.5 % BCFA grew almost as well as did those receiving the diet which included 5 % palmitic

Fatty acid composition of tissue lipids of rats 427

Table 3. Proportions of neutral lipid and of phospholipid in the total lipids of liver of rats given diets containing experimental fatty acids

Diet ~~~~~~ .

Stock (14 days) Stock (14 days); basal (14 days) 50.i Palmitic acid (14 days) 53/, Palmitic acid (14 days); basal (14 days) fS/, Phytanic acid (3 days) 5 % 3-MHD (8 days) 5 % 3-MHD (8 days): basal (14 days) 5 4;, 4-MHD (8 days) 5 % 4-MHI) (8 days); basal (14 days) 5 % BCFA (14 days) 5 % BCFA (14 days); basal (14 days) 2 . 5 % BCFA (14 days) 7 . 5 % BCFA (14 days) 10% BCFA (14 days) 150(, BCFA (3 days)

Liver

weight) (g wet

-~

7. I 8 . 0 5 .9 8 . 0 2 .0 2 .2 6 . 5 2 . I 5 .8 5 .4 8 .1 4 .9 5 .8 4 . 7 2.1

Mean values

Neutral lipid (mg g-I

wet weight)

Phospholipid

wet weight) (mg g-'

1 I . 4 10.1 12.9 9 . 8

13.0 36.0 13.7 6.7

12.0 11.7 10.0 10.5 13.0 13.9 9 .7

32. I 31.6 38.5 31.7 18.6 27.1 35.9 21.3 34.8 33.9 31.3 38.7 38.7 32.0 19.2

Percentage neutral lipid in

total lipid ~~~

26.2 24.2 25.1 23.6 41. I" 57.1n 27 .6 23.9 25.6 25.7 21.1 21.3 25.1 30.3" 33.6"

'1 Values are each significantly different ( P < O . O 5 ) from those of animals receiving the control diets.

acid. Rats given the diets which contained 10% BCFA, 3-MHD or 4-MHD barely subsisted while those receiving 15 % BCFA or 5 % phytanic acid became moribund within 3 days; the adverse effect associated with the latter diets were apparently linked to inappetance.

A substantial difference clearly exists between the growth response of rats given diets containing 4-MHD and that of rats receiving 5 % BCFA. The explanation for this discrepancy is presumably linked to the fact that though the 4-methyl configuration was by far the most common structural feature among the constituent fatty acids of the concentrate there was, nevertheless, a greater proportion, on aggregate, of the fatty acids of BCFA with even-numbered methyl substitution on carbons distal to C-4 on the acyl chain (i.e. C-6, C-8, C-10, etc.); the contribution which fatty acids with these structures make to the influence of dietary BCFA is yet to be assessed.

Effects associated with diets containing the two 3-methyl substituted acids are noteworthy because the evident toxicity of phytanic acid at the levels of 5 % inclusion in the diet was markedly more intense than that observed in rats receiving the monomethyl analogue, 3-MHD. That the latter group of rats survived longer than the animals given phytanic acid despite the fact that accumula- tion of the respective branched acids in tissue lipid was much greater in the group receiving 3-MHD indicates that the trimethyl-substituted moiety distal to the 3-methyl group in the multi-branched acid contributes significantly to the toxic properties associated with phytanic a ~ i d . ~ ~ - 2 ~ Whether this is a generalised effect arising from changes in cell membrane permeability or to more specific interactions between phytanic acid and enzymes (such as possible inhibition of desaturases) remains to be determined.

Adverse effects on growth rate resulting from ingestion of the experimental diets by the rats were readily alleviated by replacement of these diets by the basal diet. Thus, animals given the basal diet after ingestion of 3-MHD and 4-MHD, during which time their growth rates were greatly depressed, had growth rates which were similar to those of control animals.

5.2. Influence of experimental diets on the composition of tissue lipids 5.2.1. Main internal organs It is implicit in a study such as this in which unlabelled substances are given to laboratory animals that theobservations which aremade on lipid content and fatty acid composition of tissues represent

428 A. Smith st al.

Table 4. The composition of the major component fatty acids of tissue lipids of rats given diets containing experi- mental fatty acidsn (mean values as a percentage by weight of the total fatty acids)

Diet

Stock (14 days)

5 % Palniitic acid (14 days)

5 % Phytanic acid (3 days)

5;{ 3-MHD(8 days)

5 % BCFA (14 days)

Acid ~

16:O 1 8 : O 18:l 18:2 16:O 18:O 18: I 18:2 16:O 18:O 18: I 18:2

Phytanic 16:O 18:O 18: 1 18:2

3-MHD 16:O 18:O 18: 1 18:2

4-MHD 16:O 18:O 1 8 : l 18:2

BCFA

Liver ~- _______

Neutral lipid Phospholipid

27. I 30.9 6.1 40.4

30.4 10.9 21.1 7.8 31.1 27.0 9 .2 33.1

32.3 11.5 15.5 11.4 13.4 16.8 9.0 32.9 8 .9 8 . 8 5.0 6.5

46.5 21.7 6.8 9 .6 3.3 29.4 5.4 7.0 5.6 4.8

65.5 (1.7)" 33.5 (0.1) 17.2 20.2 14.1 32.0 13.6 10.2 6.9 4.6

24.2 (0.0) 17.1 (0.1) 21.4 18.1

5.6 25.8 15.3 11.9 14.7 10.3 31.8 (3.4) 21.2 (2.0)

Sciatic Heart Kidney Brain nerve

- ~~~~

15.8 16.7 20.1 30.8 20.5 17.8 21 .o 8.4 12.8 19.3 23.3 38.5 21.9 15.9 I .2 3.8 27.8 23.0 24.0 28.3 32. I 15.3 24.7 9 .0 16.9 15.3 23.6 34.9 15.2 15.7 1 .2 10.6 15.3 16. I 18.6 21 .o 20.4 18.5 22.9 11.4 9 . 9 12.5 21.9 36.5

11.5 12.1 1.6 4.4 17.0 8 . 1 0.8 0.8 5.4 10.2 20.5 20.0

10.0 13.8 21.2 10.8 5.1 9 . 1 21.6 34.7 6 .1 9.1 0.9 5.2

52.0(2.3) 32.2(2.6) 2.3(0.7) 2.5(0.1) 9.8 9 . 7 17.7 21.5

23.7 16.8 21.5 14.1 7.0 15. 1 21.1 40.9

12.4 7. I I . 4 2.4 13.4(0.9) 22.3(1.4) 2.5(1.8) 2.3(0.8) 8 .9 13.2 18.4 22.9 9.9 11.2 23.4 7.8 9 .6 13.6 22.6 31.4 8.0 13.3 1 .o 6.4

30.3(1.9) 25.0(1.9) 2.8(1.8) 13.0(4.4)

"Tables of the complete fatty acid composition of each of the tissues have been deposited with the British

b Values in parentheses refer to the proportion of BCFA remaining in the tissues of the rats given the basal diet Library and are available for consultation by quoting SUP 11032.

after the experimental diets (see Table I ) .

net values that are a resultant of the rate of assimilation of the experimental fatty acids from the gut (and their uptake into tissue lipid) and their rate of disappearance through catabolism or excretion. Differences between treatments are therefore of uncertain significance since there is no assurance that observed values represent equilibrium conditions.

However, it is evident that in relation to the lipids of the main internal organs branched acids accumulated rapidly and to a considerable extent. It appears that equilibrium between branched acids of the diet and those of tissue lipids was rapidly attained at least in relation to liver and kidney because the proportion of branched acids in these tissues in rats given BCFA increased progres- sively over the entire range of inclusion (2.5-15 %) in the diet despite the fact that animals receiving the highest level of BCFA were taken out of the experiment after only 3 days on the diet. This increase in the content of branched acids in the liver phospholipids of the rats given BCFA is presumably indicative of the rapidity with which these acids were incorporated into the constituent lipids of hepatic cells and organelles. This is probably also true of the kidney although only the total lipids of this organ were examined.

Accumulation of branched acids was particularly evident in heart. This observation is likely to be associated with the large flux of fatty acids supplied to the organ to meet energy requirements; it would also imply the existence of a degree of selectivity against the utilisation of branched acids

Fatty acid composition of tissue lipids of rats 429

compared with n-fatty acids. The likelihood that such selectivity is operative is indicated by the vastly differing degrees of accumulation of 3-MHD and 4-MHD, respectively, in the tissue lipids of rats given these acids in the same proportion in the diet and for the same length of time. In this connection, it is known that the presence of a 3-methyl substituent in a fatty acyl chain is inhibitory towards /3-oxidation and that the catabolism of such an acid only proceeds in mammalian tissue after an a-oxidation step yields the 2-methyl anal0gue.2~*2~ Furthermore, mobilisation of the 3- methyl substituted acid from tissue lipid is likely to be sterically hindered as has been shown by other w0rkers.2~

Although branched acids accumulated to a considerable extent in the lipids of liver, heart and kidney, the rate of disappearance of these acids from each of the tissues was such that after the animals were returned to the basal diet, only small proportions of branched acids remained in the tissue lipids indicating that the rate of turnover of lipids in these tissues is rapid.

If‘ the presence of undue proportions of neutral lipid in the total lipid of liver is an indication of fatty infiltration of liver tissues then it is noteworthy that, amongst all the experimental animals, only those which received phytanic acid, 3-MHD and BCFA at the 10 and 15 % level of inclusion in the diet showed signs of this condition.

5.2.2. Neural tissue lipids The content of branched acids which occurred in neural lipids as a result of the various dietary treatments was but slight in comparison with that of the lipids of the main internal organs. This is in accord with the observation that incorporation of saturated fatty acids of exogenous origin into the tissue lipids of the central and peripheral nervous system occurs only to a limited extent.2s Moreover, the rats on experiment were weanlings and had presumably passed the stage of develop- ment during which myelination of the brain is optimal.29 Also, the finding that a relatively substan- tial proportion of the branched acids deposited in the lipids of brain and sciatic nerve remained after the animals had reverted to the basal diet is indicative of the known slow rate of turnover of these acids in neural lipids. However, a more prolonged exposure of rats to diets containing BCFA might well result in branched acids being present in greater proportion in the tissues of brain and peripheral nerve than was observed in the present study. Be that as it may, the observa- tions which were made in relation to the presence of the multi-branched fatty acid, phytanic acid, in small proportions in the lipids of brain and peripheral nerve are in accord with the findings of other workers whose concerns included the possible association of phytanic acid in nerve tissue lipids with the polyneuropathy of Refsum’s Syndrome.30

Reference has already been made in the text to possible changes in membrane permeability and to enzyme activity that are consequent upon the inclusion of branched acids in the cells and organelles of the tissues examined. In this connection, the provision and availability of naturally occurring and synthetic branched acids of known configuration would afford the possibility of studying structural and functional properties of cell membranes in which normal fatty acids have been systematically replaced.

Acknowledgements The autbors wish to thank Mrs J. Walker and Mrs Y. Maxwell for expert technical assistance and Mr I. McDonald for carrying out the statistical analysis. They are also grateful to Dr A. Crossley of Unilever Research who kindly arranged facilities for the hydrogenation of the methyl esters in the preparation of the branched-acid chain fatty concentrate. The gift of palmitic acid from Price’s Chemicals Ltd, Wirral, Cheshire, is gratefully acknowledged.

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