Asia Pacific J Clin Nutr (1997) 6(1): 31-35

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Impact of saturated and trans fatty acid enriched oil blends on atherosclerosis in rabbits fed cholesterol-free diets

Kalyana Sundram¹, R Pathmanathan², KT Wong² and G Baskaran²

¹Palm Oil Research Institute of Malaysia (PORIM), Kuala Lumpur, Malaysia
²Malaysia Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

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Thirty six-male New Zealand White rabbits subdivided into four dietary groups (9 animals per group) were fed high fat (36% en), cholesterol-free diets for nine months. The dietary oil blends were formulated to contain high levels of the target fatty acids namely trans-rich (partially hydrogenated soybean oil; TRANS), cis monounsaturated-rich (rapeseed, sunflower seed oil and palm olein; MONO), palmitic-rich (palm olein; POL) and lauric-myristic rich (coconut, palm kernel and corn oils; LM). Ad libitum feeding of the rabbits resulted in normal growth throughout the nine months and no differences in the final body weights of the animals were evident at autopsy. Plasma total cholesterol was significantly elevated only by the LM enriched diet compared with all other treatments; values were comparable between the other three treatment groups. Changes in the total cholesterol were not reflected in the VLDL and LDL lipoproteins. However, HDL-cholesterol was significantly lowered by the TRANS diet compared with all other dietary groups. HDL-cholesterol was also significantly increased by the LM diet in comparison to the POL-diet. Both adipose and liver triglyceride fatty acid compositions tended to reflect the type of fatty acids fed the animals. Trans fatty acids were evident only in animals fed the trans diet and it was apparent that the trans fatty acids competed with linoleic acid for incorporation into these tissues. Increased concentrations of lauric and myristic fatty acids in the LM-fed animals were also evident. In the POL and high MONO fed rabbits, palmitic and oleic fatty acids (respectively) were concentrated in the adipose and liver. The diets, however, failed to induce severe atherosclerosis in this study. This can be explained, in part, by the lack of dietary cholesterol and the use of plant (rather than animal) proteins in our dietary formulations. The effect of these important atherosclerosis modulators in association with these fatty acids requires further evaluation.

Key words: rabbits, atherosclerosis, lipids, lipoproteins, dietary fat

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Introduction

The role of dietary fats as a determinant of plasma lipids and lipoproteins is well documented^1-3. Plasma lipid levels are influenced not only by the amount of fat consumed but by its nature as well. The degree of saturation and unsaturation, stereo isometric differences and fatty acid chain length can all determine the response of plasma cholesterol to dietary fats. The classical studies of Hegsted, Keys and their colleagues^4-6 have dissected the cholesterolaemic effects of dietary fats into their constituent fatty acid classes. They showed that saturates were twice as effective in raising plasma total cholesterol (TC) as the polyunsaturates. Among the saturated fatty acids, only those with chain lengths 12, 14 and 16 carbons (lauric, myristic and palmitic respectively) increased TC while the 18 carbon stearic acid and the monounsaturated oleic acid were recognised as neutral. These simplified fatty acid relationships and their effects on lipoprotein classes have also come under further scrutiny recently⁷. Newer data from animal^8,9 and human^10-12 studies now suggest that the cholesterolaemic effects of the saturates are not uniform. The most potent cholesterol raising fatty acid appears to be myristic acid (14:0) while palmitic acid may behave as a neutral fatty acid under certain conditions and especially when supported by sufficient amounts of linoleic acid⁷.

The fatty acid controversy has been further fuelled by the role that trans fatty acids play in the modulation of blood lipids and possibly atherosclerosis. Trans fatty acids are geometrical isomers of the unsaturated fatty acids and are produced as a result of the hydrogenation of edible oils used in margarine and shortening manufacture. Several clinical studies have shown the adverse effects of trans fatty acids^13-16; they increase TC, lipoprotein Lp(a) and reduce the protective high density lipoprotein cholesterol (HDL-C). The adverse effects of trans on lipoproteins shown in these clinical studies have additionally been supported by the epidemiological data of Willett and coworkers^17,18. A recent study of Sundram et al¹⁹ addressed an outstanding question of whether the trans fatty acids are nutritionally better or worse in these regards than the dietary saturated fatty acids they were designed to replace in solid fat products. They concluded that the negative impacts of trans elaidic acid on the lipoprotein profile of humans were worse than the saturates of chain length 12, 14 and 16 carbons.

The association between diet, plasma lipid concentrations and atherosclerosis has been well documented and reviewed²⁰. Atherosclerotic lesions in humans and in animal experimental models appear to be related to elevated plasma cholesterol and excess fat consumption. The rabbit is a frequently used experimental model for evaluating dietary fat effects and atherosclerosis. In the present study we postulated that the adverse effects of trans fatty acids compared with the saturates on lipids and lipoproteins observed in a previous human study¹⁹ may lead to the onset of atherosclerotic lesions when fed to rabbits over an extended duration. This hypothesis was therefore tested using the same fat blends as used in the above human study. The oil blends mimicked fatty acid compositions that are routinely consumed in normal human dietary situations. We however omitted the addition of dietary cholesterol in this phase of our study since dietary cholesterol significantly enhances the onset of atherosclerosis in the rabbit model.

Materials and methods

Thirty-six male New Zealand White rabbits aged 2.5 months were randomly assigned to four different dietary groups. To each group was assigned a total of 9 rabbits and dietary feeding was commenced 15 days later at the age of three months after the rabbits had been acclimatised to their new environment in the animal experimental unit. The rabbits were fed ad libitum for a total of nine months a 20% (w/w) high fat diet containing the dietary oil blends whose fatty acid composition is described in Table 1. The trans-rich oil was derived from partially hydrogenated soybean oil (35° C) which consisted of almost 39% trans fatty acids and predominated as elaidic acid. This was remixed with native soybean oil so that a final trans fatty acid content of 29.2% was achieved in the blend. The high lauric-myristic (LM) fatty acid oil was a blend of palm kernel, coconut and corn oils to reflect a high level of lauric and myristic acids matched by adequate amounts of linoleic acid from corn oil to match the cis 18:2 content in the trans blend. The monounsaturated cis 18:1 oils blend (MONO) was a blend of rapeseed oil (30%), sunflower seed oil (25%) and palm olein (45%) whereas the 16:0-rich fat was palm olein.

Table 1. Fatty acid composition of fat blends incorporated into diets (g/100 g of dietary oil)

Fatty acid	Trans-richa hydrogenated soyabean oil	MONO-richb (18:1)	Palmitic-richc (16:0)	Lauric-myristic-rich (12:0+14:0)d
SFA	17.76	23.56	44.56	60.01
8:0 10:0 12:0 14:0 16:0 18:0 20:0 22:0	ND ND ND ND 11.60 5.62 0.24 0.30	ND ND 0.19 0.46 19.08 3.14 0.34 0.35	ND ND 0.53 0.84 38.94 3.96 0.29 ND	0.75 1.91 27.08 14.61 10.84 4.58 0.23 0.10
MUFA	33.20	61.01	44.36	20.87
16:1 n-9 18:1 n-9	ND 33.20	0.14 60.87	0.11 44.25	ND 20.87
PUFA	19.83	14.9	10.89	18.83
18:2 n-6 18:3 n-3	17.60 2.23	13.63 1.27	10.74 0.15	18.60 0.23
Trans FA 18:1 n-9t 18:1 n-11t 18:1 n-13t Unid cis/trans	29.21 23.11 3.40 1.55 1.15	ND ND ND ND ND	ND ND ND ND ND	ND ND ND ND ND
P/S ratio	1.12	0.63	0.24	0.31

ND, not detected; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; P/S, polyunsaturated/saturated fatty acid ratio. (a) 70% hydrogenated soyabean oil (melting point 35^oC), 30% soyabean oil. (b) 30% rapeseed oil, 25% sunflower seed oil, 45% palm olein. (c) 100% palm olein. (d) 45% coconut oil, 15% palm kernel oil, 40% corn oil.

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Commercial rabbit diet (in pellet form) was used as the basal diet. The commercial pellets were obtained as a single production batch for the entire study duration to reduce variations in their nutrient content. This contained 2.5% fat, 16.0% protein, 48.8% carbohydrates, 18% fibre, 12.8% moisture and 1.9% vitamins, minerals (including calcium) and trace elements required for optimum maintenance of the rabbits. The protein source in this basal diet was mainly of vegetable (soy) origin. Incorporation of the oil blends into the basal rabbit pellets to increase the fat energy density of the diets was achieved as follows: 200g of each oil blend was first dissolved in 120ml of diethyl ether and this was mixed with 1.0 kg of the rabbit pellet. This was stirred in a large vessel and the diethyl ether was air-dried. The oil enriched pellets were further oven dried to remove any traces of diethyl ether in the feed. The resulting diet contained 36.4% en as fat, 50.6% en as carbohydrates and 13% en as proteins. Their fatty acid composition is given in Table 2. All animals were weighed at regular monthly intervals and monitored for their general well being and feed intakes.

Table 2. Fatty acid composition (%) and energy (%) of diets.

Fatty acid	TRANS			MONO			POL		LM
	FAC %	en %		FAC %	en %		FAC %	en %	FAC %	en %
SFA	21.0	7.6		26.2	9.4		44.2	15.9	57.4	20.7
8:0	0.3	0.1		0.3	0.1		0.3	0.1	0.8	0.3
10:0	0.1	-		0.1	-		0.1	-	1.7	0.6
12:0	1.9	0.7		2.1	0.8		2.4	0.9	25.2	9.1
14:0	0.8	0.3		1.2	0.4		1.5	0.5	13.4	4.8
16:0	12.3	4.4		18.9	6.8		36.0	13.0	11.8	4.3
18:0	5.1	1.8		3.0	1.1		3.3	1.2	4.2	1.5
20:0	0.2	-		0.3	0.1		0.3	0.1	0.2	0.1
22:0	0.3	0.1		0.3	0.1		0.3	0.1	0.1	-
MUFA	31.6	11.4		56.0	20.2		41.1	14.8	20.6	7.4
16:1n-9	ND	ND		0.4	0.1		0.1	-	0.4	0.1
18:1n-9	31.6	11.4		55.6	20.0		41.0	14.8	20.2	7.3
PUFA	21.4	7.7		17.1	6.2		13.9	5.0	20.5	7.4
18:2n-6	19.5	7.0		16.0	5.8		13.7	4.9	20.3	7.3
18:3n-3	1.9	0.7		1.1	0.4		0.2	0.1	0.2	0.1
TRANS	25.1	9.1		ND	ND		ND	ND	ND	ND
18:1n-9t	19.9	7.2		ND	ND		ND	ND	ND	ND
18:1n-11t	2.9	1.0		ND	ND		ND	ND	ND	ND
18:1n-13t	1.3	0.5		ND	ND		ND	ND	ND	ND
Unid cis/ trans	1.0	0.4		ND	ND		ND	ND	ND	ND

SFA: Saturated Fatty Acids; MUFA: Monounsaturated Fatty Acids; PUFA: Polyunsaturated Fatty Acids.

 

At the end of nine months feeding, the rabbits were fasted overnight, anaesthetised with sodium pentabarbital and had their blood collected by heart puncture. The animals were then killed by an overdose of the anaesthesia (100mg/kg body weight) and autopsied to remove the various organs of interest. The entire aorta from the aortic valve to the bifur-cation was removed quickly and placed in cold physiological saline at 4°C. The heart, kidney, liver, spleen, pancreas and lungs were removed for weight determination and samples were preserved in 10% neutral buffered formalin (pH 7.2) for histopathological examination. The aorta was opened along the mid-dorsal line to expose the intimal surface. It was then divided into arch, ascending, descending and abdominal aorta. Samples from the aorta and liver were preserved on 4% ice-cold glutaraldehyde for electron microscopy (at a later date). The aorta was examined macro-scopically; portions were pinned on a wax board and stained with Oil Red O for delineation of atheromatous deposits in the intima.

Lesions of the intimal surfaces were evaluated by microscopic examination from tissues and from constant sites from the arch, ascending and abdominal aorta, without regard to the presence or absence of gross pathology. Tissue was stained with both haematoxylin and eosin as well as with Oil Red O after cryostat section. The tissue sample at each site consisted of a transverse section of the aorta. Involvement of the aorta by atheroma was assessed by depth of lipid infiltration. Samples from other organs were stained with haematoxylin and eosin, and the general pathological alteration was documented. In addition, specific evidence of fatty change and fatty infiltration was studied.

Measurement of plasma lipids and lipoproteins

Blood collected in tubes containing EDTA (1 mg/ml blood) was used to prepare plasma for the lipid and lipoprotein analyses. Four ml of plasma was pipetted into a Beckmann 50.3 Ti ultraclear centrifuge tube and over layered with 2.0 ml NaCl solution (d20 = 1.006 g/mL). The very low density lipoproteins (VLDL) were isolated by preparative ultracentrifuge in a Beckmann 50.3 Ti rotor in a Beckmann LM8-70 ultracentrifuge. Low density and high density lipoproteins were sequentially isolated at their respective densities (d20) of (1.006<d<1.063) and (1.063<d<1.125) on a Kbr density gradient. These lipoproteins were extensively dialysed to remove their background salt densities. The cholesterol and triglycerides content of the plasma and isolated lipoproteins was analysed enzymatically on an autosampler.

Fatty acid analysis

Adipose tissue and liver were first pulverised in liquid nitrogen to facilitate the extraction of its lipids using chloroform: methanol (2:1v/v) and partitioned with salt solution as described by Folch et al²¹. The lower chloroform layer was dried under nitrogen recovered for thin layer chromatography. The total lipids thus obtained from adipose tissue and liver were aliquoted and spotted on thin-layer plates coated with silica gel G and the lipid components were separated using hexane:diethyl ether:formic acid (80:20:2 v/v/v) as solvent system in a TLC tank saturated with the solvent vapours. Lipid components were identified after spraying the plates with 7,12-dichlorofluoresceine dye in ethanol and visualised under ultraviolet light. The triglycerides in adipose tissue were thus recovered for fatty acid analysis.

Fatty acid composition of the dietary oils, diets, adipose and liver triglycerides were determined following trans-methylation of the samples using toluene-sulphuric acid¹⁹. Fatty acids were then analysed by using a Perkin Elmer Autosystem gas chromatogram (Perkin Elmer Corporation, Norwalk, CT, USA) fitted with a 100-metre capillary column (SP2560, Supelco, Belfonte, USA) and temperature programmed from 160°C to 240°C at 4°C/min.

Statistical analysis

All data were checked for their frequency distribution using the Rankitts plots. Analysis of variance and the Bonferroni Inequality test were used to test the differences between dietary treatments. Two tailed tests was performed and treatments were considered significant when p<0.05.

Results

Rabbits fed the experimental diets demonstrated normal growth throughout the nine months feeding duration. At autopsy, a diet-induced difference in body weight or in the weight of various organs was not generally evident. The diets fed to these animals had similar total fat energy densities but varied significant in the contribution of energy from the individual fatty acids (Table 3). A total of 9.1% energy as trans fatty acids was available for the TRANS diet and most of this was contributed as elaidic acid (18:1 n-9t; 7.2% en). The linoleic acid content in the LM and TRANS diets were matched so that differences in the cholesterolaemic and atherosclerotic effects of these fats, when apparent, could be ascribed to the target fatty acids (total trans in the TRANS group and lauric + myristic acids in the LM group) and not as a result of differences in the linoleic acid availability. Similarly POL was enriched in palmitic acid and oleic acids (13% en and 14.8% en) while MONO was oleic rich (20.2% en).

Table 3. Effect of dietary oil blends on rabbit lipids and lipoproteins following 9-month dietary feeding.

	TC mmol/L	TG mmol/L	VLDL-C mmol/L	LDL-C mmol/L	HDL-C mmol/L	LDL/HDL-C RATIO
TRANS	2.41 ± 0.45a	0.99 ± 0.18	0.44 ± 0.09	1.33 ± 0.38	0.64 ± 0.30a,b,c	2.08 ± 0.46a,b,c
MONO	2.44 ± 0.57b	0.86 ± 0.18	0.39 ± 0.09	1.25 ± 0.27	0.80 ± 0.38a	1.56 ± 0.39b
POL	2.52 ± 0.45c	1.01 ± 0.20	0.46 ± 0.11	1.28 ± 0.33	0.76 ± 0.21b,d	1.68 ± 0.32c
LM	2.78 ± 0.34a,b,c	0.96 ± 0.45	0.48 ± 0.20	1.32 ± 0.48	0.89 ± 0.25c,d	1.48 ± 0.37a

Values are means ± SD. (n=9 rabbits per group); Means with common superscript are significantly different (P < 0.05)

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Plasma lipids and lipoproteins were generally modulated by the diet type. Plasma total cholesterol (TC) was significantly elevated in the rabbits fed the LM diet compared to all other dietary treatments. TC values were however comparable between the TRANS, MONO and POL diets. The effect of these diets varying in their fatty acid composition was evaluated on plasma cholesterol distribution in the lipoproteins. VLDL-cholesterol was lowest in rabbits fed the MONO diet and highest in the POL group. These values did not attain significance. Similarly, LDL-cholesterol was not significantly modulated by these diets in spite of the pronounced differences in the dietary fatty acid composition. HDL-cholesterol was however lowest in the TRANS-fed animals and this lowering was significant in comparison to all other dietary treatments. HDL-cholesterol was also significantly increased by the LM diet in comparison to the POL diet. As a result of these shifts in the lipoprotein cholesterol values the ratio of LDL/HDL-cholesterol was significantly elevated by the TRANS diet in comparison to all other dietary treatments.

The adipose tissue fatty acid composition was evaluated as an index of the long-term effect of these fatty acids in these rabbits (Table 4). In general this tissue fatty acid composition reflected the type of fatty acid fed the animals. In the LM group, a significant increase in the 12:0 and 14:0 fatty acids was apparent whereas these fatty acids were present at less than 1.0% in the other dietary treatments. Palmitic acid was significantly higher in the POL fed animals while cis oleic acid was highest in the MONO treated animals. Rabbits fed the TRANS diet were characterised by the presence of trans fatty acids that were absent in all other treatments. In addition, cis 18:2 were significantly lower in the POL and MONO fed rabbits, signifying a lower availability of this essential fatty acid from the diet. However, the cis 18:2 content in the TRANS fed rabbits was obviously significantly lower than that of the LM fed rabbits although dietary availability was almost equally matched.

Table 4. Adipose tissue fatty acid composition (%) of rabbits fed experimental oil blends.

Fatty acid		TRANS	MONO	POL	LM
SFA	12:0	ND	ND	ND	4.62 ± 0.89
	14:0	0.66 ± 0.16a	0.77 ± 0.26b	0.94 ± 0.14c	7.36 ± 0.29a,b,c
	16:0	14.84 ± 0.93a,b,c	17.79 ± 1.67a,d,e	22.75 ± 0.98b,d,f	9.88 ± 1.76c,e,f
	18:0	5.05 ± 0.66	4.58 ± 0.66	5.20 ± 0.68	4.34 ± 0.46
MUFA	16:1 n-9	0.62 ± 0.30a,b,c	1.24 ±0.30a	1.10 ± 0.25b	1.31 ± 0.46c
	18:1 n-9	39.23 ± 15.68a,b,c	53.86 ± 3.09a,d,e	49.83 ± 0.79b,d,f	28.26 ±0.98c,e,f
PUFA	18:2 n-6	25.37 ± 0.66a,b,c	19.09 ± 0.98a,d	18.82 ± 0.47b,e	32.09 ± 2.03c,d,e
	18:3 n-3	1.62 ± 0.61a,b,c	1.16 ± 0.31a,d	0.84 ± 0.11b,d,e	1.17 ± 0.24c,e
TRANS	18:1 n-9t	11.31 ± 0.93	ND	ND	ND
	18:1 n-11t	0.29 ± 0.04	ND	ND	ND

Values are means ± S.D. (n=9 rabbits per group); Means with common supercript are significantly different (P < 0.05)

 

Fatty acid composition of liver triglycerides (Table 5) generally followed the trend seen in the adipose tissue and reflected the dietary availability of these fatty acids. Lauric and myristic acids were highest in the LM group, palmitic in the POL fed animals, cis oleic in the MONO fed rabbits and trans fatty acids were evident only in the TRANS group.

Table 5. Major fatty acids (%) present in liver triglycerides of rabbits fed different diets for 9 months

Fatty acid		TRANS	MONO	LM	POL
SFA	12:0	ND	0.26 ± 0.09a	2.27 ± 1.35a,b	0.20 ± 0.04b
	14:0	0.74 ± 0.46a	0.96 ± 0.10b	5.28 ± 1.80a,b,c	0.95 ± 0.15c
	16:0	18.95 ± 2.81a,b,c	29.80 ± 2.36a,d	27.59 ± 1.42b,e	31.76 ± 1.97c,d,e
	18:0	13.99 ± 2.81a,b,c	4.34 ± 0.65a	4.50 ± 1.37b	4.13 ± 1.03c
MUFA	16:1 n-9	0.50 ± 0.22a,b,c	1.31 ± 0.34a	1.17 ± 0.34b	1.15± 0.26c
	18:1 n-9	17.69 ± 2.92a,b,c	43.78 ± 2.76a,d,e	25.27 ± 2.89b,d,f	39.47 ± 1.58c,e,f
PUFA	18:2 n-6	28.93 ± 3.78a,b,c	18.97 ± 4.92a,d	31.82 ± 2.39b,d,e	20.92 ± 1.60c,e
	18:3 n-3	1.45 ± 0.88a,b,c	0.69 ± 0.16a	0.85 ± 0.19b	0.57 ± 0.14c
Trans	18:1 n-9t	4.78 ± 1.99	ND	ND	ND
	18:1 n-11t	4.49 ± 0.89	ND	ND	ND
	18:1 n-13t	1.54 ± 0.14	ND	ND	ND

Values are means ± S.D. (n=9 rabbits per group); Means with common superscript are significantly different (P < 0.05)

 

Gross pathological examination of the aorta generally showed a smooth intima with no macroscopic evidence of atheromatous lesions except in animals fed trans-rich (2/9) and POL-rich (1/9) diets. In these animals, fatty streaks and fibrous plaques were present in the arch and the ascending aorta. Histological examination of these grossly abnormal lesions showed moderate lipid infiltration with distinct elevation of the lesions, capped by a white fibrotic cap. In two other animals, (one each from trans and POL groups), there was histological evidence of mild lipid infiltration into the intima even though there was no obvious abnormality on inspection.

Most animals showed some degree of fatty change in the liver, which ranged from minimal to severe fatty change. There was no statistically significant difference between the extent of fatty change and the diet in these experimental animals. Histopathological examination of the other organs was generally unremarkable.

Discussion

In this study rabbits were fed high-fat diets continuously for the nine-month duration. Changes in blood lipids and lipoproteins resulting from these fatty acid manipulations were evident. Although the oil blends used had very different fatty acid compositions in terms of their polyunsaturated: mono-unsaturated: saturated fatty acid ratios, they nevertheless failed to elicit the expected significant differences in plasma TC and LDL-C. However HDL-C was depressed by the trans-enriched diet and the resulting LDL/HDL-cholesterol ratio was significantly lower in the trans fed animals. The same oil blends were fed to normocholesterolaemic humans in an earlier study¹⁹. In these subjects trans-enrichment in the diet resulted in adverse lipoprotein profiles (increased TC, LDL-C, Lp(a) and decreased HDL-C) which were worse than the saturates.

In an effort to delineate the effects of the different saturated fatty acids on cholesterolaemia, Hayes and Khosla⁷ have proposed that myristic acid is the most cholesterolaemic fatty acid. Its potency has been estimated to be four times that of palmitic acid that is the most abundant saturated fatty acid in the human diet. In nature, myristic acid normally coexists with lauric acid (eg. in butterfat, coconut oil and palm kernel oil) and this makes it difficult to separate the effects of lauric acid from myristic acid. In the present study the combination of lauric + myristic fatty acids did not increase TC and LDL-C compared to the other fat blends. We had increased the linoleic acid content in the LM blend and it is possible that this may have helped overcome the cholesterolaemic response of the myristic acid therein. The response between the POL (palmitic-rich) and MONO (cis 18:1-rich) was similar, a response that was also evident in the human volunteers¹⁹.

The lack of cholesterolaemic response in the present study may be partially explained on the following basis. Rabbits on low-fat, cholesterol-free semi-purified diets containing casein become hypercholesterolaemic whereas normal levels of cholesterol are maintained if the casein is replaced by isolated soy protein²². This suggests that the mechanism of hypercholesterolaemia in rabbits may be protein dependent more than fat dependent. Apart from this fact, it is well-documented²³ that rabbits are sensitive to the addition of cholesterol in their diets and will develop cholesterolaemia and atherosclerotic lesions when exposed to dietary cholesterol for even short durations. In the present study the protein source in the basal rabbit pellet used was soy based, and no dietary cholesterol was added to the diets. This may explain the low levels of plasma lipids and the lack of response on the lipoprotein profiles even though the fatty acid compositions were significantly different.

Both adipose tissue and liver triglycerides fatty acid compositions provided important insights into the utilisation of the dietary fatty acids pools in this animal atherosclerotic model. In these tissue samples trans fatty acids were evident only when animals were fed the hydrogenated oil blend. Of specific interest was the observation that the tissue linoleic and linolenic acids content were significantly lower in the TRANS than in the LM dietary group. This was in spite of the almost equal availability of these polyunsaturates in the diet from these two oil blends (TRANS, 7.7% en versus LM 7.4% en). It is now well documented that trans fatty acids increase essential fatty acid requirements^24,25 and therefore adequate levels of essential fatty acids must be present in the diet to overcome any adverse effects especially on the physiologically important biochemical pathways. In this model the lower tissue incorporation of linoleic and linolenic acids could signify that the trans had competed with these acids for some common reaction sites. The total trans energy available was high and in a situation where inadequate essential polyunsaturates are available adverse effects on some metabolic processes including lipoprotein cholesterol modulation and atherosclerosis may be expected.

It has been reported²⁶ in hamsters that dramatic hepatic changes in LDL-cholesterol metabolism occur when the diet is enriched with myristic acid. Enrichment with 18:1 n-9t (elaidic acid) however was reported to lack biochemical and physiological effects on LDL-cholesterol since in vitro ACAT cannot effectively use 18:1 n-9t for cholesterol esterification²⁷. That hypothesis concerning 18:1 n-9t fatty acid contradicts recent observations in humans^13-16 wherein LDL-cholesterol was increased and HDL-cholesterol decreased. Furthermore, CETP activity increases (thereby depressing HDL-cholesterol) when trans are incorporated in the diet²⁸. In this study LDL-cholesterol was not significantly increased by either myristic (in LM) or trans (in TRANS) fatty acids but HDL was depressed by the TRANS diet. We believe that this lack of effect on LDL-cholesterol and overall atherosclerotic index was due to the absence of dietary cholesterol and dietary casein (animal protein) in our diet formulation. The trans effect may not simply be a case of lack of activation of ACAT activity as suggested previously. The interaction of dietary cholesterol and casein along with the fatty acids of interest need to be further evaluated in a similar animal model.

Acknowledgement

The excellent technical assistance of the Nutrition Research Group staff at PORIM namely Mr. Shuhaizan, Mrs. Fatmawati, Mrs. Rosnah and Mr. Selvarajan is gratefully acknowledged. Mr. Vincent (University Malaya) assisted in the histopathological preparation of tissue samples. This study was supported by a grant from the Palm Oil Research Institue of Malaysia and we thank its Director-General for permission to publish this paper.

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Impact of saturated and trans fatty acid enriched oil blends on atherosclerosis in rabbits fed cholesterol-free diets
Kalyana Sundram, R Pathmanathan, KT Wong, G Baskaran
Asia Pacific Journal of Clinical Nutrition (1997) Volume 6, Number 1: 31-35

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References

1. Grundy SM, Denke MA. Dietary influences in serum lipids and lipoproteins. J Lipid Res 1990; 31: 1149-1168.
2. McNamara DJ, Kolb R, Parker TS et al. Heterogeneity of cholesterol homeostasis in man: responses to changes in dietary fat quality and cholesterol quantity. J Clin Invest 1987; 79: 1729-1739.
3. Gurr MI. Dietary lipids and coronary heart disease: old evidence, new perspective. Prog Lipid Res 1992; 3: 195-245.
4. Hegsted DM, McGandy RB, Myers ML, Stare FJ. Quantitative effects of dietary fat on serum cholesterol in man. Am J Clin Nutr 1965; 17: 281-295.
5. Keys A, Anderson JT, Grande F. Serum cholesterol response to changes in the diet. IV Particular saturated fatty acids in the diet. Metabolism 1965; 14: 776-787.
6. Hegsted DM. Dietary fatty acids, serum cholesterol and coronary heart disease. In: Nelson GJ, ed. Health effects of dietary fatty acids. Champaign, IL: American Oil Chemist’s Society, 1991; 50-68.
7. Hayes KC, Khosla P. Dietary fatty acid thresholds and cholesterolemia. FASEB J 1992; 6: 2600-2607.
8. Khosla P, Hayes KC. Comparison between the effects of dietary saturated (16:0), monounsaturated (18:1), and polyunsaturated (18:2) fatty acids on plasma lipoprotein metabolism in cebus and rhesus monkeys fed cholesterol-free diets. Am J Clin Nutr 1992; 55: 51-62.
9. Woolett JA, Spady DK, Dietschy JM. Saturated and unsaturated fatty acids independently regulate low density lipoprotein receptor activity and production rate. J Lipid Res 1992; 33: 77-87.
10. Sundram K, Hayes KC, Siru OH. Dietary palmitic acid results in lower serum cholesterol than does a lauric-myristic acid combination in normolipemic humans. Am J Clin Nutr 1994; 59: 841-846.
11. Bonanome A, Grundy SM. Effect of dietary stearic acid on plasma cholesterol and lipoprotein levels. N Engl J Med 1988; 319: 1244-1248.
12. Tholstrup T, Marckman P, Jespersen J, Vessby B, Jart A, Sandstorm B. Effect on blood lipids, coagulation and fibrinolysis of a fat high in myristic acid and a fat high in palmitic acid. Am J Clin Nutr 1994; 60: 919-925.
13. Mensink RP, Katan MB. Effect of dietary trans fatty acids on low-density and high-density lipoprotein cholesterol levels in healthy subjects. N Engl J Med 1990; 6: 188-194.
14. Judd JT, Clevidence BA, Muesing RA, Wittes L, Sunkin ME, Prodczasy JJ. Dietary trans fatty acids: effects on plasma lipids and lipoproteins of healthy men and women. Am J Clin Nutr 1994; 59: 861-868.
15. Nestel PJ, Noakes M, Belling B, McArthur R, Clifton PM, Janus E, Abbey M. Plasma lipoprotein lipid and Lp(a) changes with substitution of elaidic acid for oleic acid in the diet. J Lipid Res 1992; 33: 1029-1036.
16. Mensink RP, Zock PL, Katan MB, Hornstra G. Effect of dietary cis and trans fatty acids on serum lipoprotein(a) levels in healthy subjects. J Lipid Res 1992; 33: 1493-1501.
17. Ascherio A, Willett WC. Metabolic and atherogenic effects of trans fatty acids. J Int Med 1995; 238: 93-96.
18. Willett WC, Stampfer JM, Manson JE, Colditz GA, Speizer FE, Rosner BA, Sampson LA, Hennekens CH. Intake of trans fatty acids and risk of coronary heart disease among women. Lancet 1993; 341: 581-586.
19. Sundram K, Ismail A, Hayes KC, Jeyamalar R, Pathmanathan R. Trans elaidic fatty acids adversely impact lipoprotein profile relative to specific saturated fatty acids in humans. J Nutr 1997.
20. Steinberg D, Witztum JL. Lipoprotein and atherogenesis: current concepts. JAMA 1990; 264: 3047-3052.
21. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957; 226: 497-509.
22. Carroll KK. Hypercholesterolemia and atherosclerosis: effects of dietary protein. Fed Proc 1982; 41: 2792-2796.
23. Roth RI, Gaubatz JW, Gotto AM Jr., Patsch JR. Effect of cholesterol feeding on the distribution of plasma lipoproteins and on the metabolism of apolipoprotein E in the rabbit. J Lipid Res 1983; 24: 1-11.
24. Emken EA. Do trans fatty acids have adverse health consequences? In: Nelson GJ, ed. Health effects of dietary fatty acids. Champaign, IL: American Oil Chemists Society, 1991: 245-260.
25. Beyers EC, Emken EA. Metabolites of cis, trans and trans, cis isomers of linoleic acid in mice and incorporation into tissue lipids. Biochim Biophys Acta 1991; 1082: 275-284.
26. Daumerie CM, Woollett LA, Dietschy JM. Fatty acids regulate hepatic low density lipoprotein receptor activity through redistribution of intracellular cholesterol pools. Proc Natl Acad Sci USA 1992; 89: 10797-10801.
27. Sgoutas DS. Effect of geometry and position of ethylenic bond upon acyl coenzyme A-cholesterol-O-acyl-transferase. Biochemistry 1970; 9: 1826-1833.
28. Abbey M, Nestel PJ. Plasma cholesteryl ester transfer protein is increased when trans-elaidic acid is substituted for cis-oleic acid in the diet. Atherosclerosis 1994; 106: 99-107.

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