Alpha-Linolenic Acid–Rich Wheat Germ Oil Decreases Oxidative Stress and CD40 Ligand in Patients With Mild Hypercholesterolemia
Epidemiological studies have underlined that a higher intake of α-linolenic acid (18:3n-3) is inversely associated with coronary and carotid atherosclerotic disease.1 Long-chain n-3 fatty acids have been suggested to delay the atherosclerotic process through an antiinflammatory activity2 and in turn to stabilize vulnerable plaque.3 Previous studies suggested, in particular, that n-3 fatty acids may exert an antiinflammatory action by reducing oxidative stress.4 As we have previously shown that oxidative stress is implicated in upregulating CD40 ligand (CD40L), a protein with inflammatory and prothrombotic property,5,6 we tested the hypothesis that 18:3n-3 may protect against atherosclerotic disease also via inhibiting oxidative stress–mediated CD40L expression. To investigate this issue, 32 patients with hypercholesterolemia were randomly allocated, in a double blind fashion, to 2 months supplementation with 2 vegetable oils (1 tbsp/d) containing low (maize oil, 0.63%) or high (wheat germ oil, 8.6%) percentage of 18:3n-3. Before and after treatment oxidative stress and CD40L, a protein that is implicated in the progression of atherosclerotic disease,5 were measured. Also, we analyzed in vitro whether 18:3n-3 fatty acids were able to influence platelet oxidative stress and CD40L expression (for expanded Methods, see supplementary data available online at http://atvb.ahajournals.org).
There were no differences in clinical and laboratory characteristics between the 2 groups; after supplementation with both vegetable oils, no change of serum lipid profile was observed (not shown).
Maize oil supplementation did not change either soluble CD40 Ligand (sCD40L) data or 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative stress7 (not shown). Conversely, a parallel (r=0.534; P=0.03) and significant decrease of 8-OHdG (from 1.06±0.26 to 0.62±0.20 ng/mL; −41.5%, P<0.001) and sCD40L (from 223.5±32.2 to 96.5±7.1 pg/mL; −56.9%, P<0.001) was seen in wheat germ oil-treated patients. Also, platelet CD40L significantly decreased (mean fluorescence from 45.4±3.1 to 29.3±3.1; −35.4%, P<0.001) only in wheat germ oil-treated patients (Figure, panel A).
A significant increase of platelet content of linoleic acid was observed in patients given maize (n=3, +14.28%, P<0.005) or wheat germ oil (n=3, +16%, P<0.05). Conversely, a significant increase of platelet concentration of α-linolenic acid was observed only in patients given wheat germ oil (n=3, +255%, P<0,002 versus n=3, +6%, P>0.05 in patients given maize oil; Figure, panel B). Also a significant reduction in platelet arachidonic acid (n=3, −16.5%, P<0.005) was observed only in patients given wheat germ oil. Platelets from volunteers incubated with liposomes containing linoleic acid or α-linolenic acid showed a different behavior. Thus, linoleic acid demonstrated a nonsignificant decrease of CD40L platelet O2−, NADPH oxidase, and p38MAP kinase activation (data not shown); conversely platelets incubated with α-linolenic acid showed a marked reduction of platelet CD40L expression (Figure, panel C), O2− production (Figure, panel D), NADPH oxidase (Figure, panel E), and p38MAP kinase activation (Figure, panel F).
This study showed that in patients with hypercholesterolemia, wheat germ oil supplementation was associated with parallel reduction of oxidative stress and platelet CD40L expression suggesting that n-3 fatty acids downregulated CD40L via an oxidative stress–mediated mechanism. In vitro experiments showing that platelet incubation with n-3 fatty acids elicited significant decrease of platelet O2− could support such hypothesis. Moreover, platelets incubated with n-3 fatty acids showed reduced activation of NADPH oxidase and phosphorylation of p38 MAP kinase, which is a protein implicated in the activation of NADPH oxidase.8,9 The underlying mechanism cannot be fully elucidated at the moment. Accumulation of 18:3n-3 on platelet membrane could reduce the content of arachidonic acid and in turn lower NADPH oxidase activation.8 This hypothesis, which is consistent with previous data showing a key role for arachidonic acid in stimulating platelet NADPH oxidase–dependent O2− generation,10 should be investigated in the future.
Even if we cannot exclude that the n-3 antioxidant effect could not only be the cause but also the consequence of downregulation of CD40L,10 our hypothesis is consistent with an interventional study showing that supplementation with ascorbic acid was associated with platelet CD40L downregulation.11
In conclusion, we provide evidence that wheat germ oil is an important source of n-3 fatty acids, which may exert an antiatherosclerotic effect via inhibition of oxidative stress–mediated CD40L upregulation.
We are grateful to Annarita Scarca and Roberto Petruccioli for the generous gift of wheat germ oil (Germen).
Source of Funding
This study was supported in part by a grant from the University of Rome “La Sapienza” (Ateneo 2003).
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