- Email: [email protected]
Failure of conjugated linoleic acid supplementation to enhance biosynthesis of docosahexaenoic acid from α-linolenic acid in healthy human volunteers
ARTICLE IN PRESS
Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130 www.elsevier.com/locate/plefa
Failure of conjugated linoleic acid supplementation to enhance biosynthesis of docosahexaenoic acid from a-linolenic acid in healthy human volunteers N.M. Attar-Bashia, R.S. Weisingerb, D.P. Beggb, D. Lic, A.J. Sinclaird, a
School of Applied Sciences, RMIT University, Melbourne, Victoria, Australia School of Psychological Science, La Trobe University, Bundoora, Victoria, Australia c Department of Food Science and Nutrition, Zhejiang University, Hangzhou, China, d School of Exercise and Nutrition Sciences, Deakin University, 221 Burwood Highway, Burwood, Victoria 3125, Australia b
Received 23 May 2006; received in revised form 11 October 2006; accepted 7 November 2006
Abstract A rate-limiting step in docosahexaenoic acid (DHA) formation from a-linolenic acid (ALA) involves peroxisomal oxidation of 24:6n-3 to DHA. The aim of the study was to determine whether conjugated linoleic acid (CLA) would enhance conversion of ALA to DHA in humans on an ALA-supplemented diet. The subjects (n ¼ 8 per group) received daily supplementation of ALA (11 g) and either CLA (3.2 g) or placebo for 8 weeks. At baseline, 4 and 8 weeks, blood was collected for plasma fatty acid analysis and a number of physiological measures were examined. The ALA-supplemented diet increased plasma levels of ALA and eicosapentaenoic acid (EPA). The addition of CLA to the ALA diet resulted in increased plasma levels of CLA, as well as ALA and EPA. Plasma level of DHA was not increased with either the ALA alone or ALA plus CLA supplementation. The results demonstrated that CLA was not effective in enhancing DHA levels in plasma in healthy volunteers. r 2006 Elsevier Ltd. All rights reserved.
1. Introduction The effectiveness of a-linolenic acid (ALA) as a substrate for tissue eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in humans is a subject for much dispute. Diets rich in ALA do not necessarily lead to high tissue DHA levels. An early study in healthy subjects that received 30 mL linseed oil daily for 4 weeks showed that DHA in serum phospholipid (PL) was increased by 70% [1]. Also, in a long-term study, increased intake of ALA (3 g/day) resulted in more than a 20% increase in plasma DHA level in elderly subjects at 10 months, but not at 4 months [2]. In contrast, supplementation of ALA in humans, up to 15 g/day Corresponding author. Tel.: +61 3 9251 7282; fax: +61 3 9244 6017. E-mail address: [email protected] (A.J. Sinclair).
0952-3278/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2006.11.002
for 4 weeks, led to increased ALA and EPA in plasma triacylglycerol (TAG) and PL, but very little, if any, detectible increase in DHA in plasma, platelets, white blood cells and red blood cells [3–5]. Pawlosky and colleagues analysed metabolism of ALA (see Fig. 1) in adult humans that received a 1 g oral dose of penta-deuterated ALA ethyl ester (d5-18: 3n-3). The results showed that only about 0.2% of the plasma ALA was destined for synthesis of EPA, approximately 63% of the plasma EPA was accessible for production of 22:5n-3 and 37% of 22:5n-3 was available for synthesis of DHA [6]. Minimal conversion of ALA to EPA indicated that the biosynthesis of longchain n-3 PUFA from ALA is limited in healthy individuals. It has been suggested that the rate-limiting step in the formation of DHA from ALA is the final step, involving peroxisomal oxidation of 24:6n-3 to DHA [7,8]. Thus,
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
122
18:2n-6 (LA) 18:3n-3 (ALA) Δ6 Desaturase
18:3n-6 18:4n-3
20:3n-6 20:4n-3
24:4n-6 24:5n-3
24:5n-6 24:6n-3 Peroxisomal oxidation
Δ5 Desaturase
22:4n-6 22:5n-6 22:5n-3 22:6n-3 (DHA)
20:4n-6 (AA) 20:5n-3 (EPA)
Fig. 1. Pathway of conversion of LA and ALA to LCPUFA.
one strategy to enhance DHA synthesis might be to add a peroxisomal proliferating agent to the diet. Conjugated linoleic acid (CLA) is a fatty acid found in dairy products and meat from ruminants [9] and can also be produced by the alkaline isomerization of LA from vegetable oils [10]. The two main CLA isomers produced by the latter method are c9, t11-CLA and t10, c12 CLA. Unlike LA and ALA, the double bonds in these molecules are conjugated. In animals, CLA has been reported to exert beneficial effects in carcinogenesis, atherosclerosis, immune system function, lipid metabolism and body composition [11,12] and it has also been reported to act as a peroxisomal proliferator in mice [13]. Sebedio et al. [14] have reported a significant increase in liver PL DHA concentrations when rats were fed a diet supplemented with 1% CLA (10t, 12c 18:2). However, studies in rats have shown that CLA does not act as a peroxisomal proliferator and it was suggested that there might be a species difference in the effect of CLA in rats and mice [13,15,16]. The ability of CLA to act as a peroxisomal proliferator in humans, as it does in mice, is not known. The aim of this study was to determine whether supplementing healthy subjects’ diet with ALA from flaxseed oil and CLA capsules for 8 weeks would lead to an increase in DHA concentration in plasma PL, TAG and cholesterol ester (CE) fractions compared with a group supplemented with ALA from flaxseed oil and a placebo capsule (soybean oil).
2. Materials and methods 2.1. Subjects and protocol Healthy subjects aged 20–50 years were recruited through advertisements in the RMIT University newsletter, Australian Herald Sun newspaper, Manningham Leader newspaper and Victorian branch of the Australian Vegan Society website. The exclusion criteria for the study were cigarette smoking, history of bleeding
disorders, diabetes, cardiovascular disease, hypertension, hypercholesterolaemia, regular intake of antiinflammatory medications and regular eaters of fish and seafood. The subjects were randomly assigned to one of two dietary intervention groups (six males and two females per group): (1) ALA+CLA group. This group was given 20 mL flaxseed oil (11 g of ALA) (Melrose Laboratories, Melbourne Australia) plus 4 1 mL CLA capsules (3.2 g CLA) (Natural Inc., USA) per day. The dose of CLA was based on that used in human studies examining effects on weight loss [17–19]. (2) ALA+P (placebo) group. This group was given 20 mL flaxseed oil (11 g of ALA) plus 4 0.5 mL soybean oil capsules (placebo) per day (Clover Corporation, Sydney, Australia). The only placebo capsules available were 0.5 mL and it was decided to give both groups the same number of capsules rather than the same weight of oil. However, it is recognised that it would have been preferable to have 1 g placebo capsules. The fatty acid composition of the oils is shown in Table 1. Subjects received the dietary intervention for 8 weeks in addition to their habitual diet. Subjects were asked to refrain from eating fish and seafood, however if they could not avoid this they were asked to keep to a maximum of one fishmeal every fortnight. All subjects signed consent forms prior to the dietary intervention. The study was approved by the RMIT University Human Research Ethics Committee, Melbourne, Australia. 2.2. Subjects appointments Subjects were required to attend RMIT University, Department of Food Science for three visits during the Table 1 Fatty acid profile of the oils consumed by subjects during the study period (% of total fatty acids) Fatty acids
Flaxseed oila
Soybean oil capsulesb
CLA capsulesc
16:0 18:0 18:1 18:2n-6 18:3n-3 c9, t11 18:2 CLA t10,c12 18:2 CLA t9, t11 and t10, t12 CLA c9, c11 CLA
5.4 2.5 15.9 17.1 57.0 — — —
11.2 3.8 23.7 53.5 6.0 — — —
4.3 2.6 13.9 1.4 — 34.7 38.5 1.4
—
—
1.3
Values are means of analysis in duplicate. a Flaxseed oil was kindly provided by Melrose Laboratories Pty. Ltd., Victoria, Australia. b Soybean oil capsules (Placebo) were kindly provided by Clover Research Corporation, Victoria, Australia. c CLA capsules were kindly provided by Natural Inc., IL, USA.
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
study. All three visits were in the morning following an overnight fast (from 10:00 pm until the appointment); however, subjects’ water intake was not restricted. Upon arrival, the subject’s blood pressure, height, weight, waist, hip and percentage body fat were measured. Blood (30 mL) was then drawn by a certified phlebotomist, following this, breakfast was supplied. Subjects were then given flaxseed oil (and a measuring cylinder to measure the 20 mL of oil) and capsules containing either CLA or soybean oil. They were asked to bring back whatever was left of the oil and capsules to the next appointment and a fresh batch of the oil and capsules were given. 2.3. Blood processing and storing Blood was collected in EDTA tubes and kept on ice until spun in a refrigerated (4 1C) centrifuge at 2500 rpm for 10 min. Plasma was aliquotted into 1 mL Eppendorf tubes and stored in a 80 1C freezer until analysed. 2.4. Plasma total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and TAG measurements Plasma total cholesterol, HDL-C, and TAG were determined from 1 mL of plasma. LDL-C levels were calculated. Analysis was performed using a Beckman SYNCHRON LXs 20 Autoanalyzer by Monash Medical Centre, Melbourne, Australia. The analysts were blinded in regards to the sample details. 2.5. Plasma fatty acid analysis Plasma fatty acid analyses carried out by the Child Nutrition Research Unit, Flinders Medical Centre, South Australia, Australia. The analysts were blinded in regards to the sample details. Total lipids were extracted from plasma samples with chloroform: methanol. Diheptadecanoyl (C17:0)-phosphatidylcholine, triheptadecanoin (C17:0), and cholesteryl heptadecanoate (C17:0) were added to the plasma at the extraction phase as internal standards. Plasma PL, TAG and CE were separated by thin-layer chromatography (TLC). The separated lipid classes were methylated in 1% H2SO4 in methanol at 70 1C for 3 h. When cooled, the resulting methyl esters were extracted into n-heptane and transferred to gas chromatography vials containing anhydrous Na2SO4 as the dehydrating agent. Fatty acid methyl esters were separated and quantified using a Hewlett-Packard 6890 gas chromatograph equipped with a 50 m capillary column (0.33 mm) coated with BPX-70 (0.25 mm film thickness; SGE Pty. Ltd., Victoria, Australia). The injector temperature was set at 250 1C and the detector (flame ionisation) temperature
123
at 300 1C. The initial oven temperature was 140 1C and was programmed to rise to 220 1C at 5 1C/min. Helium was used as the carrier gas at a velocity of 35 cm/s. Fatty acid methyl esters were identified based on the retention time to authentic lipid standards obtained from Nuchek Prep Inc. (Elysian, MN) and where appropriate by GCMS. The assay coefficient of variation was 2–5% over 2 years. 2.6. Statistical analysis Analysis of variance (ANOVA) and, when required, Fisher’s PLSD post hoc test was used to compare the differences between the groups (Statistica, Statsoft). Repeated measure analysis was performed to compare changes between groups over time. Results reported are mean7SD in all tables and mean7SE in all graphs. P value of o0.05 was considered significant.
3. Results 3.1. Compliance Compliance was monitored by ALA and CLA levels in plasma lipids. 3.2. Physiological parameters There were no significant differences between the subjects in the ALA+CLA group and those in the ALA+P group during the 8 weeks study period in any of the physiological measures (see Table 2). Systolic blood pressure, however, was decreased at 8 weeks in the ALA+P group, compared with baseline. 3.3. Biochemical parameters There were no significant differences between the subjects in the ALA+CLA group and those in the ALA+P group during the 8 weeks study period in any of the biochemical measures (see Table 3). HDL, however, was increased at 4 weeks in the ALA+P group, compared with baseline. 3.4. Plasma polyunsaturated fatty acids (PUFA) CLA: CLA treatment was effective in increasing plasma level of CLA. In each of the three plasma fractions measured (PL, TAG and CE), at 4 and 8 weeks of the study, concentrations of c9, t11-18:2 CLA, t10, c12-CLA and total CLA were higher in the ALA+CLA group than in the ALA+P group. There was also a significant increase in the concentrations of c9, t11 18:2CLA, t10, c12-CLA and total CLA in the ALA+CLA
ARTICLE IN PRESS 124
N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
Table 2 Physiological indices at baseline, 4 weeks and 8 weeks after supplementation Physiological
Age BMI Waist/hip ratio % Body fat SBP (mmHg) DBP (mmHg)
ALA plus CLA diet group (n ¼ 8)
ALA plus placebo diet group (n ¼ 8)
Baseline
4weeks
8weeks
Baseline
4weeks
8weeks
33.178.2 24.074.3 0.870.1 30.9713.9 120.379.8 75.477.4
33.178.2 24.074.3 0.870.1 31.3714.2 115.677.9 70.878.3
33.178.2 24.174.4 0.870.1 30.9714.2 119.279.7 73.375.5
37.4712.2 25.073.8 0.870.0 32.5710.0 118.779.9 73.879.0
37.4712.2 25.073.9 0.870.1 31.6710.6 116.179.1 73.279.9
37.4712.2 24.973.8 0.870.1 31.6711.0 112.078.9 73.6710.7
Values are mean7SD; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure. po0.05, vs. baseline.
Table 3 Biochemical indices at baseline, 4 weeks and 8 weeks after supplementation Biochemical
Total cholesterol (mmol/L) Triacylglycerol (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) LDL:HDL ratio
ALA plus CLA diet group (n ¼ 8)
ALA plus placebo diet group (n ¼ 8)
Baseline
4weeks
8weeks
Baseline
4weeks
8weeks
4.670.5 1.170.3 1.570.2 2.670.4 1.770.3
4.470.6 0.970.2 1.570.3 2.670.5 1.870.6
4.670.5 0.970.2 1.570.3 2.770.4 1.970.4
4.170.6 1.070.5 1.470.3 2.370.3 1.770.4
4.270.6 0.870.3 1.570.3 2.370.4 1.670.4
4.070.8 0.970.4 1.470.3 2.270.6 1.770.4
Values are Mean7SD, HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol. po0.05, vs. baseline.
group at 4 and 8 weeks of the study compared with baseline (see Fig. 2). Omega-3 PUFA: There were no significant differences between the ALA+CLA and the ALA+P groups in the concentration of any of the measured omega-3 PUFA. Overall, subjects in both the ALA+CLA and ALA+P groups had increased concentrations of ALA (see Fig. 3) and EPA (see Fig. 4) in each of the three plasma fractions measured (PL, TAG and CE). The level of ALA and EPA achieved in plasma was similar at 4 and 8 weeks. The addition of CLA to the ALAsupplemented diet did not appear to influence the apparent conversion of ALA to DHA (see Fig. 5). Despite the increased intake of ALA and increased plasma level of ALA, EPA, and 22:5n-3 (see Table 4), the DHA level in each of the three plasma fractions measured (PL, TAG and CE) was similar to baseline value at both 4 and 8 weeks and similar between the ALA+CLA and the ALA+P groups. Omega-6 PUFA: There were no significant differences between the ALA+CLA and the ALA+P groups in the concentration of any of the measured omega-6 PUFA. Interestingly, the ALA treatment was associated with a decrease or a trend to a decrease in the plasma level of arachidonic acid (AA) (see Fig. 6). In the PL, TAG and CE fractions, relative to baseline, at 4 and/or 8 weeks, there was a decrease in the concentration of 22:5n-6 in the ALA+CLA and
ALA+P groups. In addition, in the TAG fraction, relative to baseline, at 4 weeks, there was a significant decrease in the concentration of 18:2n-6 and total omega-6 in the ALA+P group. In the CE fraction, relative to baseline, at 4 and/or 8 weeks, there was an increase in the concentration of 18:2n-6 and total omega-6 in the ALA+CLA group (see Table 4).
4. Discussion The aim of this study was to test a novel strategy to enhance DHA synthesis by the addition of a peroxisome proliferator-activated receptor alpha (PPARa) activator (CLA) to a diet rich in ALA, since the final step in the production of DHA is reported to involve peroxisomal oxidation [7,8]. The study investigated whether supplementing healthy subjects’ diet with ALA from flaxseed oil and CLA capsules would lead to an increase in DHA concentration in plasma PL, TAG and CE compared with a group supplemented with ALA from flaxseed oil and a placebo capsule (soybean oil). From the results, it can be seen that CLA was not effective in enhancing DHA levels from ALA, nor was there an increase in DHA simply by adding 20 mL flaxseed oil per day to the diet for 8 weeks. The pathway of DHA synthesis involves seven different steps. The last step is a chain shortening and
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
A
∗
14
B
∗
∗
12
8 6 4 +
+
2
ALA in PL ug/ml
CLA in PL ug/mL
10
125
10
∗
∗
∗
8 6 4
0 Baseline 4 weeks
∗
A
2
8 weeks
0
B
baseline
∗
10
8 weeks
50
40
6 +
4
+
2 0 Baseline 4 weeks
8weeks
Baseline
4weeks
8weeks
7 6
A
∗
∗
∗
∗
30 ∗
20
10
B
0
5
baseline
4
4 weeks
8 weeks
∗
∗
25
3 +
2
+
1 0 Baseline 4 weeks
8 weeks Baseline
4 weeks
8 weeks
Fig. 2. Effect of dietary A. ALA+CLA (white—&) or B. ALA+P (grey— ) on plasma c9, t11 18:2 CLA ( ) and t10,c12 18:2 CLA ( ). Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline; +po0.05 ALA+CLA vs. ALA+P.
involves peroxisomal oxidation of 24:6n-3 to DHA [7,8]. Peroxisomal proliferators exert their biological response as a result of activation of the PPAR subgroup of steroid hormone receptors and subsequently alter gene expression [20]. Activation of PPARa, the predominant PPAR subtype expressed in the liver, has been associated with hepatic peroxisomal proliferation [21,22]. A significant increase of hepatic mRNA levels of several enzymes associated with peroxisome proliferation has been found in female mice fed up to 1.5% CLA (w/w) for 6 weeks [13]. An increase in liver PL DHA concentrations was reported when rats were fed a diet supplemented with 1% 10t, 12c-CLA but not with 9c,11t-CLA or a mixture of the two isomers [14].
20 ALA in CE ug/ml
CLA in CE ug/mL
4 weeks
8
ALA in TAG ug/ml
CLA in TAG ug/mL
12
8 weeks Baseline 4 weeks
∗
∗
4 weeks
8 weeks
15
10
5
0 baseline
Fig. 3. Effect of dietary ALA+CLA (white—&) or ALA+P (grey— ) on plasma ALA. Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline.
However, it has been found that CLA does not act as a peroxisomal proliferator in rats, suggesting that there might be a species difference in the effect of CLA in rats and mice [15]. Another study reported that supplementing rats diet with 1% CLA either as 9c,11t-CLA, 10t, 12c-CLA or a mixture of the two isomers was not
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
126
30
50
∗
20
∗ ∗
40 ∗
15 10
DHA in PL ug/ml
EPA in PL ug/ml
25
30 20 10
5 0 baseline
4 weeks
0
8 weeks
5
baseline
4 weeks
8 weeks
baseline
4 weeks
8 weeks
baseline
4 weeks
8 weeks
7 ∗
∗
6
∗ DHA in TAG ug/ml
4 EPA in TAG ug/ml
∗
3 2
5 4 3 2
1 1 0 baseline
4 weeks
8 weeks
∗
∗
18 16
∗
6 5 ∗
12
DHA in CE ug/ml
EPA in CE ug/ml
14
0
10 8 6
4 3 2
4 1
2 0 baseline
4 weeks
8 weeks
0
Fig. 4. Effect of dietary ALA+CLA (white—&) or ALA+P (grey— ) on plasma EPA. Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline.
Fig. 5. Effect of dietary ALA+CLA (white—&) or ALA+P (grey— ) on plasma DHA. Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline.
effective at inducing a hepatic-peroxisome proliferation response; this was evidenced by the lack of change in the activity of characteristic enzymes such as acyl-CoA oxidase and CYP4A1 [16]. It has not been investigated whether CLA is effective in acting as a peroxisomal proliferator in humans in the same fashion it does in mice.
There are data that do not support the hypothesis for the requirement of peroxisomal oxidation in the synthesis of DHA [23]. It has been proposed that DHA is biosynthesised in mitochondria via a channelled carnitine-dependent pathway involving an omega-3 specific delta-4 desaturase, while AA, EPA and 22:5n-6 are biosynthesised by mitochondrial and microsomal
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
127
Table 4 Fatty acid concentration of selected n-3 and n-6 fatty acids in plasma phospholipid, triacylglycerol and cholesterol ester (mg/mL of plasma) Fatty acids (mg/mL)
Phospholipid
Triacylglycerol
Cholesterol ester
ALA plus CLA diet group (n ¼ 5–8)
ALA plus placebo diet group (n ¼ 5–8)
Baseline
Baseline
4 weeks
8 weeks
2.170.7
2.070.6
252.5748.6 389.4764.7 16.976.3
264.7750.2 402.1760.2 18.176.6
4 weeks 1.470.5
8 weeks 1.570.5 245.4731.3 371.5752.6 13.574.6
2.471.2 240.7736.0 388.3766.4 10.773.6
235.5751.8 356.9778.4 12.975.3
61.5721.9
72.5729.4
0.870.9 124.4728.8 142.4734.2 3.371.6
0.270.1 91.4732.6 103.6738.9 3.872.7
46.2715.5
22.2711.2
32.3720.1
0.170.1 452.4779.9 508.7786.4 0.470.2
0.170.1 469.3768.6 525.7767.6 0.470.2
0.270.1 390.4756.8 449.0769.4 0.270.1
0.170.1 416.5791.4 468.17101.8 0.370.1
0.17 0.1 422.2781.9 475.1793.6 0.370.2
38.1715.1
37.6716.8
15.576.3
30.4712.9
29.6711.5
22:5n-6 18:2n-6 Total n-6 22:5n-3
3.171.6 228.8765.7 389.7793.9 11.074.7
Total n-3
56.8718.8
22:5n-6 18:2n-6 Total n-6 22:5n-3
1.070.7 106.1770.4 122.8777.7 2.771.3
0.570.3 94.1745.2 104.9747.7 3.771.0
Total n-3
16.779.0
30.379.8
22:5n-6 18:2n-6 Total n-6 22:5n-3
0.270.1 374.4770.0 437.5771.0 0.270.1
Total n-3
15.676.8
79.7727.1
84.4728.1 0.570.3 106.7757.7 119.9762.4 4.872.4
74.6722.7 0.270.2 119.1741.3 132.7744.9 4.171.3 42.8720.3
Values are mean7SD. po0.05, vs. baseline; +po0.05, ALA+CLA vs. ALA+P.
systems [24]. It is not known whether the inability of CLA to increase DHA synthesis from ALA is due to CLA not being an active peroxisomal proliferator in humans (at the dose used in this study) or if the last step in DHA synthesis does not involve peroxisomal proliferation. Given that the concentration of CLA in the various plasma fractions was increased by 2.5–9.5 fold, if CLA was an active peroxisomal proliferator in humans a change in DHA level should have been produced assuming DHA synthesis requires peroxisomal proliferation. In this study, subjects were supplemented with 20 mL of flaxseed oil (11 g ALA) per day for 8 weeks. There was a significant increase in ALA and EPA in plasma PL, TAG and CE with no increase in plasma DHA. This is consistent with previous studies which have shown that feeding high ALA diets (up to 15 g/day) in 4-week studies led to significant increases in ALA, EPA and 22:5n-3 in plasma TAG and PL, and no significant increase in DHA in plasma, platelets, white and red blood cells [3,4]. A physiological compartmental analysis of ALA metabolism in eight adult humans, where subjects received a 1 g oral dose of an isotope tracer of ALA, found there was very limited conversion of ALA to EPA [6]. The results of the current study indicate that the supplementation of CLA in the diet does not increase this conversion. This is consistent with the recently reported finding that a 1.2 g dose of CLA (either isomer) does not affect metabolism of U-[(13)C]-LA or U-[(13)C]-ALA to longer chain PUFA over 168 h period in humans [25].
Recently it has been reported, in studies using 13CALA, that young women have a greater capacity than young men to convert ALA to DHA [26,27]. In the present study, although only two of the eight subjects in each group were women, no difference was observed between men and women in the apparent capacity to synthesise DHA over the 8 weeks of the study. There are a number of reasons for the relatively poor efficiency of the conversion of ALA to EPA and DHA [28]. First, a substantial proportion of the ALA is either diverted to b-oxidation or found distributed throughout all major tissue lipid pools (adipose, carcass, skin) as ALA. Second, LA is a competitive inhibitor of the metabolism of ALA to 18:4n-3, a precursor of longchain n-3 PUFA. Diets rich in LA decrease the expression of the hepatic delta-6 desaturase (compared with a diet rich in oleic acid (OA)) [29], which presumably also reduces the possibility of conversion of ALA to 18:4n-3 and 24:5n-3 to 24:6n-3. In this study, c9, t11 18:2 CLA and t10, c12 18:2 CLA were found in highest concentrations and proportions in the TAG, then in the PL and least in the CE lipids fractions. The concentrations and proportions of c9, t11 18:2 CLA were higher than t10, c12 18:2 CLA in all the three lipid classes. Previous human and in vitro studies have shown higher amounts of c9, t11 18:2 CLA concentrations than t10, c12 18:2 CLA in human plasma and tissues in culture [30,31]. The importance of this observation is not yet established, as research has not yet determined if this is a result of increased metabolism of the t10, c12 18:2 CLA or increased
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
128
140 120
AA in PL ug/ml
100
∗
∗
∗
80 60 40 20 0 baseline
4 weeks
8 weeks
12
AA in TAG ug/ml
10 8
∗
∗
∗
6 4 2 0 baseline
4 weeks
8 weeks
baseline
4 weeks
8 weeks
60
AA in CE ug/ml
50 40 30 20 10 0
Fig. 6. Effect of dietary ALA+CLA (white—&) or ALA+P (grey— ) on plasma AA. Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline.
incorporation of c9, t11 18:2 CLA into the lipid fractions. The n-3 PUFA showed different partitioning into the three lipid classes. The concentrations and proportions of ALA were highest in the TAG fraction followed by CE and PL lipid fractions. 22:5n-3 and DHA had highest concentrations and proportions in the PL lipid fraction followed by TAG and CE. Previous studies have shown this trend of n-3 PUFA partitioning
into lipid classes [26,27]. The increased ALA intake in this study led to a decrease in the plasma PL, TAG and CE n-6/n-3 ratio and LA/ALA ratio in the two groups. This observation has been reported in previous studies in humans and animals [32,33]. There was no significant change in plasma TC, LDLC, HDL-C and TAG in the ALA+CLA group and the ALA+P group. This is consistent with Benito et al. [30], who found that CLA supplementation (3.9 g/day) for 63 days did not alter the levels of plasma TC, LDL-C, HDL-C and TAG. Conversely, however, Cunnane et al. [34] reported a significant decrease in plasma TC and LDL-C in 10 healthy adults following 4 weeks of ALA supplementation with flaxseed-based muffins (9 g of ALA/day) compared with a low ALA-control diet. The effects of dietary OA, LA and ALA on plasma lipid metabolism have also been examined. There were significant reductions in the mean concentrations of plasma TC (18%), LDL-C (22%) and VLDL-C (41%) after experimental diets compared with a mixed-fat diet [35]. The dietary interventions in the current study had no effect on BMI, waist/hip ratio and % body fat over the 8 weeks of the study. CLA supplementation to animals has been shown to decrease body fat and increase lean body mass [36–39]. Decreased body fat mass was found in 47 overweight or moderately obese men and women after being supplemented with 3.4 or 6.8 g of CLA per day for 12 weeks [40]. In another study, supplementing healthy men and women diet with 4.5 g CLA per day resulted in a significant decrease in the proportion of body fat when compared with the control group [41]. However, others found no effect of 3 g of CLA per day on body weight of healthy normal weight women [42]. It is possible that the different effects of CLA on body fat in human subjects are due to the different amounts and isomers of CLA being supplemented in each study. CLA-induced body changes in animals have been linked to increased lipolysis in adipocytes and enhanced fatty acid oxidation in both adipocytes and skeletal muscle cells [37,43]. The blood pressure of all subjects in the present study was in the normal healthy range. Systolic, but not diastolic, blood pressure was decreased in the ALA+P group and neither systolic nor diastolic blood pressure was changed in the ALA+CLA subjects. Berry and Hirsch [44] studied the relationship between adipose tissue fatty acids and blood pressure in 399 male subjects. It was found that an absolute 1% increase in adipose tissue ALA was associated with a decrease of 5 mmHg in the systolic, diastolic, and composite mean arterial blood pressure [44]. At present, the aetiology of this change in blood pressure is unknown. This study demonstrated that consumption of the ALA-supplemented diet resulted in increased concentrations of ALA, EPA and 22:5n-3 in plasma at 4 and 8
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
weeks. Clearly, the rate-limiting step in the conversion of ALA to DHA lies somewhere after the conversion to 22:5n-3. In support of the involvement of peroxisomes in the synthesis of DHA, it has recently been reported that rats treated for up to 14 days with clofibrate, an agonist of the PPARa, showed a marked increased in myocardial DHA proportion and a decrease in the proportion of AA [45]. In the heart, activation of PPARa increases a variety of enzymes including peroxisomal oxidation of fatty acids. The results of this study showed that CLA was not effective in enhancing DHA synthesis from ALA in healthy human subjects, although the plasma level of CLA was clearly increased. Other studies have shown that the synthesis of DHA from ALA is slow in healthy individuals. There were significant changes in plasma fatty acid composition in the CLA supplemented group, and no changes to serum lipids or body fat in either of the two groups.
Acknowledgements The support of grants from the Australian Research Council (DP0346830 to RSW and AJS), the National Health and Medical Research Centre (217011 to RSW, 350313 to RSW and AJS), the Australian Health Management Group and the National Heart Foundation of Australia is gratefully acknowledged. The supply of the CLA capsules by Dr. Efi Farmakalidis and flaxseed oil by Melrose Laboratories is also gratefully acknowledged.
References [1] H.J. Mest, J. Beitz, I. Heinroth, H.U. Block, W. Forster, The influence of linseed oil diet on fatty acid pattern in phospholipids and thromboxane formation in platelets in man, Klin. Wochenschr. 61 (1983) 187–191. [2] O. Ezaki, M. Takahashi, T. Shigematsu, K. Shimamura, J. Kimura, H. Ezaki, T. Gotoh, Long-term effects of dietary alphalinolenic acid from perilla oil on serum fatty acids composition and on the risk factors of coronary heart disease in Japanese elderly subjects, J. Nutr. Sci. Vitaminol. (Tokyo) 45 (1999) 759– 772. [3] H. Gerster, Can adults adequately convert alpha-linolenic acid (18:3n-3) to eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3)?, Int. J. Vitam. Nutr. Res. 68 (1998) 159–173. [4] E. Mantzioris, M.J. James, R.A. Gibson, L.G. Cleland, Dietary substitution with an alpha-linolenic acid-rich vegetable oil increases eicosapentaenoic acid concentrations in tissues, Am. J. Clin. Nutr. 59 (1994) 1304–1309. [5] D. Li, A. Sinclair, A. Wilson, S. Nakkote, F. Kelly, L. Abedin, N. Mann, A. Turner, The effect of dietary alpha-linolenic acid on thrombotic risk factors in vegetarian men, Am. J. Clin. Nutr. 69 (1999) 872–882.
129
[6] R.J. Pawlosky, J.R. Hibbeln, J.A. Novotny, N. Salem Jr., Physiological compartmental analysis of alpha-linolenic acid metabolism in adult humans, J. Lipid Res. 42 (2001) 1257–1265. [7] A. Voss, M. Reinhart, S. Sankarappa, H. Sprecher, The metabolism of 7,10,13,16,19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase, J. Biol. Chem. 266 (1991) 19995–20000. [8] S.A. Moore, E. Hurt, E. Yoder, H. Sprecher, A.A. Spector, Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid, J. Lipid Res. 36 (1995) 2433–2443. [9] S.F. Chin, W. Liu, J.M. Storkson, L.A. Ha, M.W. Pariza, Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens, J. Food Compos. Anal. 5 (1992) 185–197. [10] P.L.J. Nichols, S.F. Herb, R.W. Riemenschneider, Isomers of conjugated fatty acids. I. Alkali-isomerized linoleic acid, J. Am. Chem. Soc. 73 (1951) 247–252. [11] H.B. MacDonald, Conjugated linoleic acid and disease prevention: a review of current knowledge, J. Am. Coll. Nutr. 19 (2000) 111S–118S. [12] M.A. Belury, Conjugated dienoic linoleate: a polyunsaturated fatty acid with unique chemoprotective properties, Nutr. Rev. 53 (1995) 83–89. [13] M.A. Belury, A. Kempa-Steczko, Conjugated linoleic acid modulates hepatic lipid composition in mice, Lipids 32 (1997) 199–204. [14] J.L. Sebedio, E. Angioni, J.M. Chardigny, S. Gregoire, P. Juaneda, O. Berdeaux, The effect of conjugated linoleic acid isomers on fatty acid profiles of liver and adipose tissues and their conversion to isomers of 16:2 and 18:3 conjugated fatty acids in rats, Lipids 36 (2001) 575–582. [15] P.A. Jones, L.J. Lea, R.U. Pendlington, Investigation of the potential of conjugated linoleic acid (Cla) to cause peroxisome proliferation in rats, Food Chem. Toxicol. 37 (1999) 1119–1125. [16] J.C. Martin, S. Gregoire, M.H. Siess, M. Genty, J.M. Chardigny, O. Berdeaux, P. Juaneda, J.L. Sebedio, Effects of conjugated linoleic acid isomers on lipid-metabolizing enzymes in male rats, Lipids 35 (2000) 91–98. [17] T.M. Larsen, S. Toubro, O. Gudmundsen, A. Astrup, Conjugated linoleic acid supplementation for 1 y does not prevent weight or body fat regain, Am. J. Clin. Nutr. 83 (2006) 606–612. [18] M.M. Kamphuis, M.P. Lejeune, W.H. Saris, M.S. WesterterpPlantenga, The effect of conjugated linoleic acid supplementation after weight loss on body weight regain, body composition, and resting metabolic rate in overweight subjects, Int. J. Obes. Relat. Metab. Disord. 27 (2003) 840–847. [19] A.C. Watras, A.C. Buchholz, R.N. Close, Z. Zhang, D.A. Schoeller, The role of conjugated linoleic acid in reducing body fat and preventing holiday weight gain, Int. J. Obes. (London) (2006). [20] N. Latruffe, J. Vamecq, Peroxisome proliferators and peroxisome proliferator activated receptors (PPARs) as regulators of lipid metabolism, Biochimie 79 (1997) 81–94. [21] S.S. Lee, T. Pineau, J. Drago, E.J. Lee, J.W. Owens, D.L. Kroetz, P.M. Fernandez-Salguero, H.F.J.G. Westphal, Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators, Mol. Cell Biol. 15 (1995). [22] F.J. Gonzalez, Recent update on the PPAR alpha-null mouse, Biochimie 79 (1997) 139–144. [23] J.P. Infante, V.A. Huszagh, Zellweger syndrome knockout mouse models challenge putative peroxisomal beta-oxidation involvement in docosahexaenoic acid (22:6n-3) biosynthesis, Mol. Genet. Metab. 72 (2001) 1–7.
ARTICLE IN PRESS 130
N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
[24] J.P. Infante, V.A. Huszagh, On the molecular etiology of decreased arachidonic (20:4n-6), docosapentaenoic (22:5n-6) and docosahexaenoic (22:6n-3) acids in Zellweger syndrome and other peroxisomal disorders, Mol. Cell Biochem. 168 (1997) 101–115. [25] A.M. Turpeinen, S. Barlund, R. Freese, P. Lawrence, J.T. Brenna, Effects of conjugated linoleic acid on linoleic and linolenic acid metabolism in man, Br. J. Nutr. 95 (2006) 727–733. [26] G.C. Burdge, A.E. Jones, S.A. Wootton, Eicosapentaenoic and docosapentaenoic acids are the principal products of alpha-linolenic acid metabolism in young men, Br. J. Nutr. 88 (2002) 355–363. [27] G.C. Burdge, S.A. Wootton, Conversion of alpha-linolenic acid to eicosapentaenoic, docosapentaenoic and docosahexaenoic acids in young women, Br. J. Nutr. 88 (2002) 411–420. [28] A.J. Sinclair, N.M. Attar-Bashi, D. Li, What is the role of alphalinolenic acid for mammals?, Lipids 37 (2002) 1113–1123. [29] H.P. Cho, M.T. Nakamura, S.D. Clarke, Cloning, expression, and nutritional regulation of the mammalian delta-6 desaturase, J. Biol. Chem. 274 (1999) 471–477. [30] P. Benito, G.J. Nelson, D.S. Kelley, G. Bartolini, P.C. Schmidt, V. Simon, The effect of conjugated linoleic acid on plasma lipoproteins and tissue fatty acid composition in humans, Lipids 36 (2001) 229–236. [31] M. Evans, Y. Park, M. Pariza, L. Curtis, B. Kuebler, M. McIntosh, Trans-10,cis-12 conjugated linoleic acid reduces triglyceride content while differentially affecting peroxisome proliferator activated receptor gamma2 and aP2 expression in 3T3-L1 preadipocytes, Lipids 36 (2001) 1223–1232. [32] L. Abedin, E.L. Lien, A.J. Vingrys, A.J. Sinclair, The effects of dietary alpha-linolenic acid compared with docosahexaenoic acid on brain, retina, liver, and heart in the guinea pig, Lipids 34 (1999) 475–482. [33] Z. Fu, A.J. Sinclair, Increased alpha-linolenic acid intake increases tissue alpha-linolenic acid content and apparent oxidation with little effect on tissue docosahexaenoic acid in the guinea pig, Lipids 35 (2000) 395–400. [34] S.C. Cunnane, M.J. Hamadeh, A.C. Leide, L.U. Thompson, T.M. Wolever, D.J. Jenkins, Nutritional attributes of traditional flaxseed in healthy young adults, Am. J. Clin. Nutr. 61 (1994).
[35] J.K. Chan, V.M. Bruce, B.E. McDonald, Dietary alpha-linolenic acid is as effective as oleic acid and linoleic acid in lowering blood cholesterol in normolipidemic men, Am. J. Clin. Nutr. 53 (1991) 1230–1234. [36] J.P. DeLany, F. Blohm, A.A. Truett, J.A. Scimeca, D.B. West, Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake, Am. J. Physiol. 276 (1999) R1172–R1179. [37] Y. Park, K.J. Albright, J.M. Storkson, W. Liu, M.E. Cook, M.W. Pariza, Changes in body composition in mice during feeding and withdrawal of conjugated linoleic acid, Lipids 34 (1999) 243–248. [38] G.I. Stangl, Conjugated linoleic acids exhibit a strong fat-to-lean partitioning effect, reduce serum VLDL lipids and redistribute tissue lipids in food-restricted rats, J. Nutr. 130 (2000) 1140–1146. [39] E. Ostrowska, M. Muralitharan, R.F. Cross, D.E. Bauman, F.R. Dunshea, Dietary conjugated linoleic acids increase lean tissue and decrease fat deposition in growing pigs, J. Nutr. 129 (1999) 2037–2042. [40] H. Blankson, J.A. Stakkestad, H. Fagertun, E. Thom, J. Wadstein, O. Gudmundsen, Conjugated linoleic acid reduces body fat mass in overweight and obese humans, J. Nutr. 130 (2000) 2943–2948. [41] A. Smedman, B. Vessby, Conjugated linoleic acid supplementation in humans—metabolic effects, Lipids 36 (2001) 773–781. [42] K.L. Zambell, N.L. Keim, M.D. Van Loan, B. Gale, P. Benito, D.S. Kelley, G.J. Nelson, Conjugated linoleic acid supplementation in humans: effects on body composition and energy expenditure, Lipids 35 (2000) 777–782. [43] M.W. Pariza, Animal studies: summary, gaps, and future research, Am. J. Clin. Nutr. 66 (1997) 1539S–1540S. [44] E.M. Berry, J. Hirsch, Does dietary linolenic acid influence blood pressure?, Am. J. Clin. Nutr. 44 (1986) 336–340. [45] Q. Tian, F.A. Grzemski, S. Panagiotopoulos, J.T. Ahokas, Peroxisome proliferator-activated receptor alpha agonist, clofibrate, has profound influence on myocardial fatty acid composition, Chem. Biol. Interact. 160 (2006) 241–251.
Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130 www.elsevier.com/locate/plefa
Failure of conjugated linoleic acid supplementation to enhance biosynthesis of docosahexaenoic acid from a-linolenic acid in healthy human volunteers N.M. Attar-Bashia, R.S. Weisingerb, D.P. Beggb, D. Lic, A.J. Sinclaird, a
School of Applied Sciences, RMIT University, Melbourne, Victoria, Australia School of Psychological Science, La Trobe University, Bundoora, Victoria, Australia c Department of Food Science and Nutrition, Zhejiang University, Hangzhou, China, d School of Exercise and Nutrition Sciences, Deakin University, 221 Burwood Highway, Burwood, Victoria 3125, Australia b
Received 23 May 2006; received in revised form 11 October 2006; accepted 7 November 2006
Abstract A rate-limiting step in docosahexaenoic acid (DHA) formation from a-linolenic acid (ALA) involves peroxisomal oxidation of 24:6n-3 to DHA. The aim of the study was to determine whether conjugated linoleic acid (CLA) would enhance conversion of ALA to DHA in humans on an ALA-supplemented diet. The subjects (n ¼ 8 per group) received daily supplementation of ALA (11 g) and either CLA (3.2 g) or placebo for 8 weeks. At baseline, 4 and 8 weeks, blood was collected for plasma fatty acid analysis and a number of physiological measures were examined. The ALA-supplemented diet increased plasma levels of ALA and eicosapentaenoic acid (EPA). The addition of CLA to the ALA diet resulted in increased plasma levels of CLA, as well as ALA and EPA. Plasma level of DHA was not increased with either the ALA alone or ALA plus CLA supplementation. The results demonstrated that CLA was not effective in enhancing DHA levels in plasma in healthy volunteers. r 2006 Elsevier Ltd. All rights reserved.
1. Introduction The effectiveness of a-linolenic acid (ALA) as a substrate for tissue eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in humans is a subject for much dispute. Diets rich in ALA do not necessarily lead to high tissue DHA levels. An early study in healthy subjects that received 30 mL linseed oil daily for 4 weeks showed that DHA in serum phospholipid (PL) was increased by 70% [1]. Also, in a long-term study, increased intake of ALA (3 g/day) resulted in more than a 20% increase in plasma DHA level in elderly subjects at 10 months, but not at 4 months [2]. In contrast, supplementation of ALA in humans, up to 15 g/day Corresponding author. Tel.: +61 3 9251 7282; fax: +61 3 9244 6017. E-mail address: [email protected] (A.J. Sinclair).
0952-3278/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2006.11.002
for 4 weeks, led to increased ALA and EPA in plasma triacylglycerol (TAG) and PL, but very little, if any, detectible increase in DHA in plasma, platelets, white blood cells and red blood cells [3–5]. Pawlosky and colleagues analysed metabolism of ALA (see Fig. 1) in adult humans that received a 1 g oral dose of penta-deuterated ALA ethyl ester (d5-18: 3n-3). The results showed that only about 0.2% of the plasma ALA was destined for synthesis of EPA, approximately 63% of the plasma EPA was accessible for production of 22:5n-3 and 37% of 22:5n-3 was available for synthesis of DHA [6]. Minimal conversion of ALA to EPA indicated that the biosynthesis of longchain n-3 PUFA from ALA is limited in healthy individuals. It has been suggested that the rate-limiting step in the formation of DHA from ALA is the final step, involving peroxisomal oxidation of 24:6n-3 to DHA [7,8]. Thus,
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
122
18:2n-6 (LA) 18:3n-3 (ALA) Δ6 Desaturase
18:3n-6 18:4n-3
20:3n-6 20:4n-3
24:4n-6 24:5n-3
24:5n-6 24:6n-3 Peroxisomal oxidation
Δ5 Desaturase
22:4n-6 22:5n-6 22:5n-3 22:6n-3 (DHA)
20:4n-6 (AA) 20:5n-3 (EPA)
Fig. 1. Pathway of conversion of LA and ALA to LCPUFA.
one strategy to enhance DHA synthesis might be to add a peroxisomal proliferating agent to the diet. Conjugated linoleic acid (CLA) is a fatty acid found in dairy products and meat from ruminants [9] and can also be produced by the alkaline isomerization of LA from vegetable oils [10]. The two main CLA isomers produced by the latter method are c9, t11-CLA and t10, c12 CLA. Unlike LA and ALA, the double bonds in these molecules are conjugated. In animals, CLA has been reported to exert beneficial effects in carcinogenesis, atherosclerosis, immune system function, lipid metabolism and body composition [11,12] and it has also been reported to act as a peroxisomal proliferator in mice [13]. Sebedio et al. [14] have reported a significant increase in liver PL DHA concentrations when rats were fed a diet supplemented with 1% CLA (10t, 12c 18:2). However, studies in rats have shown that CLA does not act as a peroxisomal proliferator and it was suggested that there might be a species difference in the effect of CLA in rats and mice [13,15,16]. The ability of CLA to act as a peroxisomal proliferator in humans, as it does in mice, is not known. The aim of this study was to determine whether supplementing healthy subjects’ diet with ALA from flaxseed oil and CLA capsules for 8 weeks would lead to an increase in DHA concentration in plasma PL, TAG and cholesterol ester (CE) fractions compared with a group supplemented with ALA from flaxseed oil and a placebo capsule (soybean oil).
2. Materials and methods 2.1. Subjects and protocol Healthy subjects aged 20–50 years were recruited through advertisements in the RMIT University newsletter, Australian Herald Sun newspaper, Manningham Leader newspaper and Victorian branch of the Australian Vegan Society website. The exclusion criteria for the study were cigarette smoking, history of bleeding
disorders, diabetes, cardiovascular disease, hypertension, hypercholesterolaemia, regular intake of antiinflammatory medications and regular eaters of fish and seafood. The subjects were randomly assigned to one of two dietary intervention groups (six males and two females per group): (1) ALA+CLA group. This group was given 20 mL flaxseed oil (11 g of ALA) (Melrose Laboratories, Melbourne Australia) plus 4 1 mL CLA capsules (3.2 g CLA) (Natural Inc., USA) per day. The dose of CLA was based on that used in human studies examining effects on weight loss [17–19]. (2) ALA+P (placebo) group. This group was given 20 mL flaxseed oil (11 g of ALA) plus 4 0.5 mL soybean oil capsules (placebo) per day (Clover Corporation, Sydney, Australia). The only placebo capsules available were 0.5 mL and it was decided to give both groups the same number of capsules rather than the same weight of oil. However, it is recognised that it would have been preferable to have 1 g placebo capsules. The fatty acid composition of the oils is shown in Table 1. Subjects received the dietary intervention for 8 weeks in addition to their habitual diet. Subjects were asked to refrain from eating fish and seafood, however if they could not avoid this they were asked to keep to a maximum of one fishmeal every fortnight. All subjects signed consent forms prior to the dietary intervention. The study was approved by the RMIT University Human Research Ethics Committee, Melbourne, Australia. 2.2. Subjects appointments Subjects were required to attend RMIT University, Department of Food Science for three visits during the Table 1 Fatty acid profile of the oils consumed by subjects during the study period (% of total fatty acids) Fatty acids
Flaxseed oila
Soybean oil capsulesb
CLA capsulesc
16:0 18:0 18:1 18:2n-6 18:3n-3 c9, t11 18:2 CLA t10,c12 18:2 CLA t9, t11 and t10, t12 CLA c9, c11 CLA
5.4 2.5 15.9 17.1 57.0 — — —
11.2 3.8 23.7 53.5 6.0 — — —
4.3 2.6 13.9 1.4 — 34.7 38.5 1.4
—
—
1.3
Values are means of analysis in duplicate. a Flaxseed oil was kindly provided by Melrose Laboratories Pty. Ltd., Victoria, Australia. b Soybean oil capsules (Placebo) were kindly provided by Clover Research Corporation, Victoria, Australia. c CLA capsules were kindly provided by Natural Inc., IL, USA.
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
study. All three visits were in the morning following an overnight fast (from 10:00 pm until the appointment); however, subjects’ water intake was not restricted. Upon arrival, the subject’s blood pressure, height, weight, waist, hip and percentage body fat were measured. Blood (30 mL) was then drawn by a certified phlebotomist, following this, breakfast was supplied. Subjects were then given flaxseed oil (and a measuring cylinder to measure the 20 mL of oil) and capsules containing either CLA or soybean oil. They were asked to bring back whatever was left of the oil and capsules to the next appointment and a fresh batch of the oil and capsules were given. 2.3. Blood processing and storing Blood was collected in EDTA tubes and kept on ice until spun in a refrigerated (4 1C) centrifuge at 2500 rpm for 10 min. Plasma was aliquotted into 1 mL Eppendorf tubes and stored in a 80 1C freezer until analysed. 2.4. Plasma total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and TAG measurements Plasma total cholesterol, HDL-C, and TAG were determined from 1 mL of plasma. LDL-C levels were calculated. Analysis was performed using a Beckman SYNCHRON LXs 20 Autoanalyzer by Monash Medical Centre, Melbourne, Australia. The analysts were blinded in regards to the sample details. 2.5. Plasma fatty acid analysis Plasma fatty acid analyses carried out by the Child Nutrition Research Unit, Flinders Medical Centre, South Australia, Australia. The analysts were blinded in regards to the sample details. Total lipids were extracted from plasma samples with chloroform: methanol. Diheptadecanoyl (C17:0)-phosphatidylcholine, triheptadecanoin (C17:0), and cholesteryl heptadecanoate (C17:0) were added to the plasma at the extraction phase as internal standards. Plasma PL, TAG and CE were separated by thin-layer chromatography (TLC). The separated lipid classes were methylated in 1% H2SO4 in methanol at 70 1C for 3 h. When cooled, the resulting methyl esters were extracted into n-heptane and transferred to gas chromatography vials containing anhydrous Na2SO4 as the dehydrating agent. Fatty acid methyl esters were separated and quantified using a Hewlett-Packard 6890 gas chromatograph equipped with a 50 m capillary column (0.33 mm) coated with BPX-70 (0.25 mm film thickness; SGE Pty. Ltd., Victoria, Australia). The injector temperature was set at 250 1C and the detector (flame ionisation) temperature
123
at 300 1C. The initial oven temperature was 140 1C and was programmed to rise to 220 1C at 5 1C/min. Helium was used as the carrier gas at a velocity of 35 cm/s. Fatty acid methyl esters were identified based on the retention time to authentic lipid standards obtained from Nuchek Prep Inc. (Elysian, MN) and where appropriate by GCMS. The assay coefficient of variation was 2–5% over 2 years. 2.6. Statistical analysis Analysis of variance (ANOVA) and, when required, Fisher’s PLSD post hoc test was used to compare the differences between the groups (Statistica, Statsoft). Repeated measure analysis was performed to compare changes between groups over time. Results reported are mean7SD in all tables and mean7SE in all graphs. P value of o0.05 was considered significant.
3. Results 3.1. Compliance Compliance was monitored by ALA and CLA levels in plasma lipids. 3.2. Physiological parameters There were no significant differences between the subjects in the ALA+CLA group and those in the ALA+P group during the 8 weeks study period in any of the physiological measures (see Table 2). Systolic blood pressure, however, was decreased at 8 weeks in the ALA+P group, compared with baseline. 3.3. Biochemical parameters There were no significant differences between the subjects in the ALA+CLA group and those in the ALA+P group during the 8 weeks study period in any of the biochemical measures (see Table 3). HDL, however, was increased at 4 weeks in the ALA+P group, compared with baseline. 3.4. Plasma polyunsaturated fatty acids (PUFA) CLA: CLA treatment was effective in increasing plasma level of CLA. In each of the three plasma fractions measured (PL, TAG and CE), at 4 and 8 weeks of the study, concentrations of c9, t11-18:2 CLA, t10, c12-CLA and total CLA were higher in the ALA+CLA group than in the ALA+P group. There was also a significant increase in the concentrations of c9, t11 18:2CLA, t10, c12-CLA and total CLA in the ALA+CLA
ARTICLE IN PRESS 124
N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
Table 2 Physiological indices at baseline, 4 weeks and 8 weeks after supplementation Physiological
Age BMI Waist/hip ratio % Body fat SBP (mmHg) DBP (mmHg)
ALA plus CLA diet group (n ¼ 8)
ALA plus placebo diet group (n ¼ 8)
Baseline
4weeks
8weeks
Baseline
4weeks
8weeks
33.178.2 24.074.3 0.870.1 30.9713.9 120.379.8 75.477.4
33.178.2 24.074.3 0.870.1 31.3714.2 115.677.9 70.878.3
33.178.2 24.174.4 0.870.1 30.9714.2 119.279.7 73.375.5
37.4712.2 25.073.8 0.870.0 32.5710.0 118.779.9 73.879.0
37.4712.2 25.073.9 0.870.1 31.6710.6 116.179.1 73.279.9
37.4712.2 24.973.8 0.870.1 31.6711.0 112.078.9 73.6710.7
Values are mean7SD; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure. po0.05, vs. baseline.
Table 3 Biochemical indices at baseline, 4 weeks and 8 weeks after supplementation Biochemical
Total cholesterol (mmol/L) Triacylglycerol (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) LDL:HDL ratio
ALA plus CLA diet group (n ¼ 8)
ALA plus placebo diet group (n ¼ 8)
Baseline
4weeks
8weeks
Baseline
4weeks
8weeks
4.670.5 1.170.3 1.570.2 2.670.4 1.770.3
4.470.6 0.970.2 1.570.3 2.670.5 1.870.6
4.670.5 0.970.2 1.570.3 2.770.4 1.970.4
4.170.6 1.070.5 1.470.3 2.370.3 1.770.4
4.270.6 0.870.3 1.570.3 2.370.4 1.670.4
4.070.8 0.970.4 1.470.3 2.270.6 1.770.4
Values are Mean7SD, HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol. po0.05, vs. baseline.
group at 4 and 8 weeks of the study compared with baseline (see Fig. 2). Omega-3 PUFA: There were no significant differences between the ALA+CLA and the ALA+P groups in the concentration of any of the measured omega-3 PUFA. Overall, subjects in both the ALA+CLA and ALA+P groups had increased concentrations of ALA (see Fig. 3) and EPA (see Fig. 4) in each of the three plasma fractions measured (PL, TAG and CE). The level of ALA and EPA achieved in plasma was similar at 4 and 8 weeks. The addition of CLA to the ALAsupplemented diet did not appear to influence the apparent conversion of ALA to DHA (see Fig. 5). Despite the increased intake of ALA and increased plasma level of ALA, EPA, and 22:5n-3 (see Table 4), the DHA level in each of the three plasma fractions measured (PL, TAG and CE) was similar to baseline value at both 4 and 8 weeks and similar between the ALA+CLA and the ALA+P groups. Omega-6 PUFA: There were no significant differences between the ALA+CLA and the ALA+P groups in the concentration of any of the measured omega-6 PUFA. Interestingly, the ALA treatment was associated with a decrease or a trend to a decrease in the plasma level of arachidonic acid (AA) (see Fig. 6). In the PL, TAG and CE fractions, relative to baseline, at 4 and/or 8 weeks, there was a decrease in the concentration of 22:5n-6 in the ALA+CLA and
ALA+P groups. In addition, in the TAG fraction, relative to baseline, at 4 weeks, there was a significant decrease in the concentration of 18:2n-6 and total omega-6 in the ALA+P group. In the CE fraction, relative to baseline, at 4 and/or 8 weeks, there was an increase in the concentration of 18:2n-6 and total omega-6 in the ALA+CLA group (see Table 4).
4. Discussion The aim of this study was to test a novel strategy to enhance DHA synthesis by the addition of a peroxisome proliferator-activated receptor alpha (PPARa) activator (CLA) to a diet rich in ALA, since the final step in the production of DHA is reported to involve peroxisomal oxidation [7,8]. The study investigated whether supplementing healthy subjects’ diet with ALA from flaxseed oil and CLA capsules would lead to an increase in DHA concentration in plasma PL, TAG and CE compared with a group supplemented with ALA from flaxseed oil and a placebo capsule (soybean oil). From the results, it can be seen that CLA was not effective in enhancing DHA levels from ALA, nor was there an increase in DHA simply by adding 20 mL flaxseed oil per day to the diet for 8 weeks. The pathway of DHA synthesis involves seven different steps. The last step is a chain shortening and
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
A
∗
14
B
∗
∗
12
8 6 4 +
+
2
ALA in PL ug/ml
CLA in PL ug/mL
10
125
10
∗
∗
∗
8 6 4
0 Baseline 4 weeks
∗
A
2
8 weeks
0
B
baseline
∗
10
8 weeks
50
40
6 +
4
+
2 0 Baseline 4 weeks
8weeks
Baseline
4weeks
8weeks
7 6
A
∗
∗
∗
∗
30 ∗
20
10
B
0
5
baseline
4
4 weeks
8 weeks
∗
∗
25
3 +
2
+
1 0 Baseline 4 weeks
8 weeks Baseline
4 weeks
8 weeks
Fig. 2. Effect of dietary A. ALA+CLA (white—&) or B. ALA+P (grey— ) on plasma c9, t11 18:2 CLA ( ) and t10,c12 18:2 CLA ( ). Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline; +po0.05 ALA+CLA vs. ALA+P.
involves peroxisomal oxidation of 24:6n-3 to DHA [7,8]. Peroxisomal proliferators exert their biological response as a result of activation of the PPAR subgroup of steroid hormone receptors and subsequently alter gene expression [20]. Activation of PPARa, the predominant PPAR subtype expressed in the liver, has been associated with hepatic peroxisomal proliferation [21,22]. A significant increase of hepatic mRNA levels of several enzymes associated with peroxisome proliferation has been found in female mice fed up to 1.5% CLA (w/w) for 6 weeks [13]. An increase in liver PL DHA concentrations was reported when rats were fed a diet supplemented with 1% 10t, 12c-CLA but not with 9c,11t-CLA or a mixture of the two isomers [14].
20 ALA in CE ug/ml
CLA in CE ug/mL
4 weeks
8
ALA in TAG ug/ml
CLA in TAG ug/mL
12
8 weeks Baseline 4 weeks
∗
∗
4 weeks
8 weeks
15
10
5
0 baseline
Fig. 3. Effect of dietary ALA+CLA (white—&) or ALA+P (grey— ) on plasma ALA. Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline.
However, it has been found that CLA does not act as a peroxisomal proliferator in rats, suggesting that there might be a species difference in the effect of CLA in rats and mice [15]. Another study reported that supplementing rats diet with 1% CLA either as 9c,11t-CLA, 10t, 12c-CLA or a mixture of the two isomers was not
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
126
30
50
∗
20
∗ ∗
40 ∗
15 10
DHA in PL ug/ml
EPA in PL ug/ml
25
30 20 10
5 0 baseline
4 weeks
0
8 weeks
5
baseline
4 weeks
8 weeks
baseline
4 weeks
8 weeks
baseline
4 weeks
8 weeks
7 ∗
∗
6
∗ DHA in TAG ug/ml
4 EPA in TAG ug/ml
∗
3 2
5 4 3 2
1 1 0 baseline
4 weeks
8 weeks
∗
∗
18 16
∗
6 5 ∗
12
DHA in CE ug/ml
EPA in CE ug/ml
14
0
10 8 6
4 3 2
4 1
2 0 baseline
4 weeks
8 weeks
0
Fig. 4. Effect of dietary ALA+CLA (white—&) or ALA+P (grey— ) on plasma EPA. Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline.
Fig. 5. Effect of dietary ALA+CLA (white—&) or ALA+P (grey— ) on plasma DHA. Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline.
effective at inducing a hepatic-peroxisome proliferation response; this was evidenced by the lack of change in the activity of characteristic enzymes such as acyl-CoA oxidase and CYP4A1 [16]. It has not been investigated whether CLA is effective in acting as a peroxisomal proliferator in humans in the same fashion it does in mice.
There are data that do not support the hypothesis for the requirement of peroxisomal oxidation in the synthesis of DHA [23]. It has been proposed that DHA is biosynthesised in mitochondria via a channelled carnitine-dependent pathway involving an omega-3 specific delta-4 desaturase, while AA, EPA and 22:5n-6 are biosynthesised by mitochondrial and microsomal
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
127
Table 4 Fatty acid concentration of selected n-3 and n-6 fatty acids in plasma phospholipid, triacylglycerol and cholesterol ester (mg/mL of plasma) Fatty acids (mg/mL)
Phospholipid
Triacylglycerol
Cholesterol ester
ALA plus CLA diet group (n ¼ 5–8)
ALA plus placebo diet group (n ¼ 5–8)
Baseline
Baseline
4 weeks
8 weeks
2.170.7
2.070.6
252.5748.6 389.4764.7 16.976.3
264.7750.2 402.1760.2 18.176.6
4 weeks 1.470.5
8 weeks 1.570.5 245.4731.3 371.5752.6 13.574.6
2.471.2 240.7736.0 388.3766.4 10.773.6
235.5751.8 356.9778.4 12.975.3
61.5721.9
72.5729.4
0.870.9 124.4728.8 142.4734.2 3.371.6
0.270.1 91.4732.6 103.6738.9 3.872.7
46.2715.5
22.2711.2
32.3720.1
0.170.1 452.4779.9 508.7786.4 0.470.2
0.170.1 469.3768.6 525.7767.6 0.470.2
0.270.1 390.4756.8 449.0769.4 0.270.1
0.170.1 416.5791.4 468.17101.8 0.370.1
0.17 0.1 422.2781.9 475.1793.6 0.370.2
38.1715.1
37.6716.8
15.576.3
30.4712.9
29.6711.5
22:5n-6 18:2n-6 Total n-6 22:5n-3
3.171.6 228.8765.7 389.7793.9 11.074.7
Total n-3
56.8718.8
22:5n-6 18:2n-6 Total n-6 22:5n-3
1.070.7 106.1770.4 122.8777.7 2.771.3
0.570.3 94.1745.2 104.9747.7 3.771.0
Total n-3
16.779.0
30.379.8
22:5n-6 18:2n-6 Total n-6 22:5n-3
0.270.1 374.4770.0 437.5771.0 0.270.1
Total n-3
15.676.8
79.7727.1
84.4728.1 0.570.3 106.7757.7 119.9762.4 4.872.4
74.6722.7 0.270.2 119.1741.3 132.7744.9 4.171.3 42.8720.3
Values are mean7SD. po0.05, vs. baseline; +po0.05, ALA+CLA vs. ALA+P.
systems [24]. It is not known whether the inability of CLA to increase DHA synthesis from ALA is due to CLA not being an active peroxisomal proliferator in humans (at the dose used in this study) or if the last step in DHA synthesis does not involve peroxisomal proliferation. Given that the concentration of CLA in the various plasma fractions was increased by 2.5–9.5 fold, if CLA was an active peroxisomal proliferator in humans a change in DHA level should have been produced assuming DHA synthesis requires peroxisomal proliferation. In this study, subjects were supplemented with 20 mL of flaxseed oil (11 g ALA) per day for 8 weeks. There was a significant increase in ALA and EPA in plasma PL, TAG and CE with no increase in plasma DHA. This is consistent with previous studies which have shown that feeding high ALA diets (up to 15 g/day) in 4-week studies led to significant increases in ALA, EPA and 22:5n-3 in plasma TAG and PL, and no significant increase in DHA in plasma, platelets, white and red blood cells [3,4]. A physiological compartmental analysis of ALA metabolism in eight adult humans, where subjects received a 1 g oral dose of an isotope tracer of ALA, found there was very limited conversion of ALA to EPA [6]. The results of the current study indicate that the supplementation of CLA in the diet does not increase this conversion. This is consistent with the recently reported finding that a 1.2 g dose of CLA (either isomer) does not affect metabolism of U-[(13)C]-LA or U-[(13)C]-ALA to longer chain PUFA over 168 h period in humans [25].
Recently it has been reported, in studies using 13CALA, that young women have a greater capacity than young men to convert ALA to DHA [26,27]. In the present study, although only two of the eight subjects in each group were women, no difference was observed between men and women in the apparent capacity to synthesise DHA over the 8 weeks of the study. There are a number of reasons for the relatively poor efficiency of the conversion of ALA to EPA and DHA [28]. First, a substantial proportion of the ALA is either diverted to b-oxidation or found distributed throughout all major tissue lipid pools (adipose, carcass, skin) as ALA. Second, LA is a competitive inhibitor of the metabolism of ALA to 18:4n-3, a precursor of longchain n-3 PUFA. Diets rich in LA decrease the expression of the hepatic delta-6 desaturase (compared with a diet rich in oleic acid (OA)) [29], which presumably also reduces the possibility of conversion of ALA to 18:4n-3 and 24:5n-3 to 24:6n-3. In this study, c9, t11 18:2 CLA and t10, c12 18:2 CLA were found in highest concentrations and proportions in the TAG, then in the PL and least in the CE lipids fractions. The concentrations and proportions of c9, t11 18:2 CLA were higher than t10, c12 18:2 CLA in all the three lipid classes. Previous human and in vitro studies have shown higher amounts of c9, t11 18:2 CLA concentrations than t10, c12 18:2 CLA in human plasma and tissues in culture [30,31]. The importance of this observation is not yet established, as research has not yet determined if this is a result of increased metabolism of the t10, c12 18:2 CLA or increased
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
128
140 120
AA in PL ug/ml
100
∗
∗
∗
80 60 40 20 0 baseline
4 weeks
8 weeks
12
AA in TAG ug/ml
10 8
∗
∗
∗
6 4 2 0 baseline
4 weeks
8 weeks
baseline
4 weeks
8 weeks
60
AA in CE ug/ml
50 40 30 20 10 0
Fig. 6. Effect of dietary ALA+CLA (white—&) or ALA+P (grey— ) on plasma AA. Statistical analysis by two-way ANOVA repeated measures on one variable and subsequent LSD test; *po0.05 vs. baseline.
incorporation of c9, t11 18:2 CLA into the lipid fractions. The n-3 PUFA showed different partitioning into the three lipid classes. The concentrations and proportions of ALA were highest in the TAG fraction followed by CE and PL lipid fractions. 22:5n-3 and DHA had highest concentrations and proportions in the PL lipid fraction followed by TAG and CE. Previous studies have shown this trend of n-3 PUFA partitioning
into lipid classes [26,27]. The increased ALA intake in this study led to a decrease in the plasma PL, TAG and CE n-6/n-3 ratio and LA/ALA ratio in the two groups. This observation has been reported in previous studies in humans and animals [32,33]. There was no significant change in plasma TC, LDLC, HDL-C and TAG in the ALA+CLA group and the ALA+P group. This is consistent with Benito et al. [30], who found that CLA supplementation (3.9 g/day) for 63 days did not alter the levels of plasma TC, LDL-C, HDL-C and TAG. Conversely, however, Cunnane et al. [34] reported a significant decrease in plasma TC and LDL-C in 10 healthy adults following 4 weeks of ALA supplementation with flaxseed-based muffins (9 g of ALA/day) compared with a low ALA-control diet. The effects of dietary OA, LA and ALA on plasma lipid metabolism have also been examined. There were significant reductions in the mean concentrations of plasma TC (18%), LDL-C (22%) and VLDL-C (41%) after experimental diets compared with a mixed-fat diet [35]. The dietary interventions in the current study had no effect on BMI, waist/hip ratio and % body fat over the 8 weeks of the study. CLA supplementation to animals has been shown to decrease body fat and increase lean body mass [36–39]. Decreased body fat mass was found in 47 overweight or moderately obese men and women after being supplemented with 3.4 or 6.8 g of CLA per day for 12 weeks [40]. In another study, supplementing healthy men and women diet with 4.5 g CLA per day resulted in a significant decrease in the proportion of body fat when compared with the control group [41]. However, others found no effect of 3 g of CLA per day on body weight of healthy normal weight women [42]. It is possible that the different effects of CLA on body fat in human subjects are due to the different amounts and isomers of CLA being supplemented in each study. CLA-induced body changes in animals have been linked to increased lipolysis in adipocytes and enhanced fatty acid oxidation in both adipocytes and skeletal muscle cells [37,43]. The blood pressure of all subjects in the present study was in the normal healthy range. Systolic, but not diastolic, blood pressure was decreased in the ALA+P group and neither systolic nor diastolic blood pressure was changed in the ALA+CLA subjects. Berry and Hirsch [44] studied the relationship between adipose tissue fatty acids and blood pressure in 399 male subjects. It was found that an absolute 1% increase in adipose tissue ALA was associated with a decrease of 5 mmHg in the systolic, diastolic, and composite mean arterial blood pressure [44]. At present, the aetiology of this change in blood pressure is unknown. This study demonstrated that consumption of the ALA-supplemented diet resulted in increased concentrations of ALA, EPA and 22:5n-3 in plasma at 4 and 8
ARTICLE IN PRESS N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
weeks. Clearly, the rate-limiting step in the conversion of ALA to DHA lies somewhere after the conversion to 22:5n-3. In support of the involvement of peroxisomes in the synthesis of DHA, it has recently been reported that rats treated for up to 14 days with clofibrate, an agonist of the PPARa, showed a marked increased in myocardial DHA proportion and a decrease in the proportion of AA [45]. In the heart, activation of PPARa increases a variety of enzymes including peroxisomal oxidation of fatty acids. The results of this study showed that CLA was not effective in enhancing DHA synthesis from ALA in healthy human subjects, although the plasma level of CLA was clearly increased. Other studies have shown that the synthesis of DHA from ALA is slow in healthy individuals. There were significant changes in plasma fatty acid composition in the CLA supplemented group, and no changes to serum lipids or body fat in either of the two groups.
Acknowledgements The support of grants from the Australian Research Council (DP0346830 to RSW and AJS), the National Health and Medical Research Centre (217011 to RSW, 350313 to RSW and AJS), the Australian Health Management Group and the National Heart Foundation of Australia is gratefully acknowledged. The supply of the CLA capsules by Dr. Efi Farmakalidis and flaxseed oil by Melrose Laboratories is also gratefully acknowledged.
References [1] H.J. Mest, J. Beitz, I. Heinroth, H.U. Block, W. Forster, The influence of linseed oil diet on fatty acid pattern in phospholipids and thromboxane formation in platelets in man, Klin. Wochenschr. 61 (1983) 187–191. [2] O. Ezaki, M. Takahashi, T. Shigematsu, K. Shimamura, J. Kimura, H. Ezaki, T. Gotoh, Long-term effects of dietary alphalinolenic acid from perilla oil on serum fatty acids composition and on the risk factors of coronary heart disease in Japanese elderly subjects, J. Nutr. Sci. Vitaminol. (Tokyo) 45 (1999) 759– 772. [3] H. Gerster, Can adults adequately convert alpha-linolenic acid (18:3n-3) to eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3)?, Int. J. Vitam. Nutr. Res. 68 (1998) 159–173. [4] E. Mantzioris, M.J. James, R.A. Gibson, L.G. Cleland, Dietary substitution with an alpha-linolenic acid-rich vegetable oil increases eicosapentaenoic acid concentrations in tissues, Am. J. Clin. Nutr. 59 (1994) 1304–1309. [5] D. Li, A. Sinclair, A. Wilson, S. Nakkote, F. Kelly, L. Abedin, N. Mann, A. Turner, The effect of dietary alpha-linolenic acid on thrombotic risk factors in vegetarian men, Am. J. Clin. Nutr. 69 (1999) 872–882.
129
[6] R.J. Pawlosky, J.R. Hibbeln, J.A. Novotny, N. Salem Jr., Physiological compartmental analysis of alpha-linolenic acid metabolism in adult humans, J. Lipid Res. 42 (2001) 1257–1265. [7] A. Voss, M. Reinhart, S. Sankarappa, H. Sprecher, The metabolism of 7,10,13,16,19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase, J. Biol. Chem. 266 (1991) 19995–20000. [8] S.A. Moore, E. Hurt, E. Yoder, H. Sprecher, A.A. Spector, Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid, J. Lipid Res. 36 (1995) 2433–2443. [9] S.F. Chin, W. Liu, J.M. Storkson, L.A. Ha, M.W. Pariza, Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens, J. Food Compos. Anal. 5 (1992) 185–197. [10] P.L.J. Nichols, S.F. Herb, R.W. Riemenschneider, Isomers of conjugated fatty acids. I. Alkali-isomerized linoleic acid, J. Am. Chem. Soc. 73 (1951) 247–252. [11] H.B. MacDonald, Conjugated linoleic acid and disease prevention: a review of current knowledge, J. Am. Coll. Nutr. 19 (2000) 111S–118S. [12] M.A. Belury, Conjugated dienoic linoleate: a polyunsaturated fatty acid with unique chemoprotective properties, Nutr. Rev. 53 (1995) 83–89. [13] M.A. Belury, A. Kempa-Steczko, Conjugated linoleic acid modulates hepatic lipid composition in mice, Lipids 32 (1997) 199–204. [14] J.L. Sebedio, E. Angioni, J.M. Chardigny, S. Gregoire, P. Juaneda, O. Berdeaux, The effect of conjugated linoleic acid isomers on fatty acid profiles of liver and adipose tissues and their conversion to isomers of 16:2 and 18:3 conjugated fatty acids in rats, Lipids 36 (2001) 575–582. [15] P.A. Jones, L.J. Lea, R.U. Pendlington, Investigation of the potential of conjugated linoleic acid (Cla) to cause peroxisome proliferation in rats, Food Chem. Toxicol. 37 (1999) 1119–1125. [16] J.C. Martin, S. Gregoire, M.H. Siess, M. Genty, J.M. Chardigny, O. Berdeaux, P. Juaneda, J.L. Sebedio, Effects of conjugated linoleic acid isomers on lipid-metabolizing enzymes in male rats, Lipids 35 (2000) 91–98. [17] T.M. Larsen, S. Toubro, O. Gudmundsen, A. Astrup, Conjugated linoleic acid supplementation for 1 y does not prevent weight or body fat regain, Am. J. Clin. Nutr. 83 (2006) 606–612. [18] M.M. Kamphuis, M.P. Lejeune, W.H. Saris, M.S. WesterterpPlantenga, The effect of conjugated linoleic acid supplementation after weight loss on body weight regain, body composition, and resting metabolic rate in overweight subjects, Int. J. Obes. Relat. Metab. Disord. 27 (2003) 840–847. [19] A.C. Watras, A.C. Buchholz, R.N. Close, Z. Zhang, D.A. Schoeller, The role of conjugated linoleic acid in reducing body fat and preventing holiday weight gain, Int. J. Obes. (London) (2006). [20] N. Latruffe, J. Vamecq, Peroxisome proliferators and peroxisome proliferator activated receptors (PPARs) as regulators of lipid metabolism, Biochimie 79 (1997) 81–94. [21] S.S. Lee, T. Pineau, J. Drago, E.J. Lee, J.W. Owens, D.L. Kroetz, P.M. Fernandez-Salguero, H.F.J.G. Westphal, Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators, Mol. Cell Biol. 15 (1995). [22] F.J. Gonzalez, Recent update on the PPAR alpha-null mouse, Biochimie 79 (1997) 139–144. [23] J.P. Infante, V.A. Huszagh, Zellweger syndrome knockout mouse models challenge putative peroxisomal beta-oxidation involvement in docosahexaenoic acid (22:6n-3) biosynthesis, Mol. Genet. Metab. 72 (2001) 1–7.
ARTICLE IN PRESS 130
N.M. Attar-Bashi et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 121–130
[24] J.P. Infante, V.A. Huszagh, On the molecular etiology of decreased arachidonic (20:4n-6), docosapentaenoic (22:5n-6) and docosahexaenoic (22:6n-3) acids in Zellweger syndrome and other peroxisomal disorders, Mol. Cell Biochem. 168 (1997) 101–115. [25] A.M. Turpeinen, S. Barlund, R. Freese, P. Lawrence, J.T. Brenna, Effects of conjugated linoleic acid on linoleic and linolenic acid metabolism in man, Br. J. Nutr. 95 (2006) 727–733. [26] G.C. Burdge, A.E. Jones, S.A. Wootton, Eicosapentaenoic and docosapentaenoic acids are the principal products of alpha-linolenic acid metabolism in young men, Br. J. Nutr. 88 (2002) 355–363. [27] G.C. Burdge, S.A. Wootton, Conversion of alpha-linolenic acid to eicosapentaenoic, docosapentaenoic and docosahexaenoic acids in young women, Br. J. Nutr. 88 (2002) 411–420. [28] A.J. Sinclair, N.M. Attar-Bashi, D. Li, What is the role of alphalinolenic acid for mammals?, Lipids 37 (2002) 1113–1123. [29] H.P. Cho, M.T. Nakamura, S.D. Clarke, Cloning, expression, and nutritional regulation of the mammalian delta-6 desaturase, J. Biol. Chem. 274 (1999) 471–477. [30] P. Benito, G.J. Nelson, D.S. Kelley, G. Bartolini, P.C. Schmidt, V. Simon, The effect of conjugated linoleic acid on plasma lipoproteins and tissue fatty acid composition in humans, Lipids 36 (2001) 229–236. [31] M. Evans, Y. Park, M. Pariza, L. Curtis, B. Kuebler, M. McIntosh, Trans-10,cis-12 conjugated linoleic acid reduces triglyceride content while differentially affecting peroxisome proliferator activated receptor gamma2 and aP2 expression in 3T3-L1 preadipocytes, Lipids 36 (2001) 1223–1232. [32] L. Abedin, E.L. Lien, A.J. Vingrys, A.J. Sinclair, The effects of dietary alpha-linolenic acid compared with docosahexaenoic acid on brain, retina, liver, and heart in the guinea pig, Lipids 34 (1999) 475–482. [33] Z. Fu, A.J. Sinclair, Increased alpha-linolenic acid intake increases tissue alpha-linolenic acid content and apparent oxidation with little effect on tissue docosahexaenoic acid in the guinea pig, Lipids 35 (2000) 395–400. [34] S.C. Cunnane, M.J. Hamadeh, A.C. Leide, L.U. Thompson, T.M. Wolever, D.J. Jenkins, Nutritional attributes of traditional flaxseed in healthy young adults, Am. J. Clin. Nutr. 61 (1994).
[35] J.K. Chan, V.M. Bruce, B.E. McDonald, Dietary alpha-linolenic acid is as effective as oleic acid and linoleic acid in lowering blood cholesterol in normolipidemic men, Am. J. Clin. Nutr. 53 (1991) 1230–1234. [36] J.P. DeLany, F. Blohm, A.A. Truett, J.A. Scimeca, D.B. West, Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake, Am. J. Physiol. 276 (1999) R1172–R1179. [37] Y. Park, K.J. Albright, J.M. Storkson, W. Liu, M.E. Cook, M.W. Pariza, Changes in body composition in mice during feeding and withdrawal of conjugated linoleic acid, Lipids 34 (1999) 243–248. [38] G.I. Stangl, Conjugated linoleic acids exhibit a strong fat-to-lean partitioning effect, reduce serum VLDL lipids and redistribute tissue lipids in food-restricted rats, J. Nutr. 130 (2000) 1140–1146. [39] E. Ostrowska, M. Muralitharan, R.F. Cross, D.E. Bauman, F.R. Dunshea, Dietary conjugated linoleic acids increase lean tissue and decrease fat deposition in growing pigs, J. Nutr. 129 (1999) 2037–2042. [40] H. Blankson, J.A. Stakkestad, H. Fagertun, E. Thom, J. Wadstein, O. Gudmundsen, Conjugated linoleic acid reduces body fat mass in overweight and obese humans, J. Nutr. 130 (2000) 2943–2948. [41] A. Smedman, B. Vessby, Conjugated linoleic acid supplementation in humans—metabolic effects, Lipids 36 (2001) 773–781. [42] K.L. Zambell, N.L. Keim, M.D. Van Loan, B. Gale, P. Benito, D.S. Kelley, G.J. Nelson, Conjugated linoleic acid supplementation in humans: effects on body composition and energy expenditure, Lipids 35 (2000) 777–782. [43] M.W. Pariza, Animal studies: summary, gaps, and future research, Am. J. Clin. Nutr. 66 (1997) 1539S–1540S. [44] E.M. Berry, J. Hirsch, Does dietary linolenic acid influence blood pressure?, Am. J. Clin. Nutr. 44 (1986) 336–340. [45] Q. Tian, F.A. Grzemski, S. Panagiotopoulos, J.T. Ahokas, Peroxisome proliferator-activated receptor alpha agonist, clofibrate, has profound influence on myocardial fatty acid composition, Chem. Biol. Interact. 160 (2006) 241–251.