Effects of milk enriched with ω-3 fatty acid, oleic acid and folic acid in patients with metabolic syndrome

ARTICLE IN PRESS Clinical Nutrition (2006) 25, 581–587

http://intl.elsevierhealth.com/journals/clnu

ORIGINAL ARTICLE

Effects of milk enriched with x-3 fatty acid, oleic acid and folic acid in patients with metabolic syndrome Pedro Benitoa,b,, Javier Caballerob,c, Jesus Morenoa,b, ˜ozb,c, Gemma Rojob,d, Carmen Gutie ´rrez-Alca ´ntaraa,b, Carmen Mun Sara Garciab,d, Federico C. Soriguerb,d a

Department of Endocrinology, Reina Sofı´a Hospital, Co ´rdoba University, Spain Department of Edocrinology, Carlos Haya Hospital, Ma´laga, Spain c Department of Biochemistry, Reina Sofı´a Hospital, Co ´rdoba University, Spain d Methodological Investigation Unit, Reina Sofı´a Hospital, Co ´rdoba University, Spain b

Received 1 September 2005; accepted 12 December 2005

KEYWORDS o-3 fatty acids; Oleic acid; Folic acid; Cardiovascular risk factors; Metabolic syndrome; n-3 fatty acids; Fortified milk; Folate

Summary Background & aims: Patients with metabolic syndrome (MS) have increased cardiovascular risk factors. Dietary modifications mainly polyunsatturated fatty acids intake, can improve them. The present study was performed to assess the effects of enriched milk with o-3 and oleic fatty acids, folic acid and vitamin E, in these patients. Methods: We performed a randomized, placebo-controlled and open clinical trial, among 72 patients with MS for 3 months. Thirty-six of them consumed 500 cm3 per day of semi-skimmed milk (control group), and the others consumed 500 cm3 per day of enriched milk (test group). Daily supplements in this group were 5.7 g of oleic acid, 0.2 g of o-3 fatty acid, 150 mg of folic acid and 7.5 mg of vitamin E. Serum for total and HDL cholesterol, triglycerol, Apo B, glucose, insulin, hs-CRP, homocysteine and fatty acids contents in serum phospholipids, was obtained at the beginning and at the end of the study. LDL cholesterol was calculated by Friedewald formula. Results: Four patients in the test group, and two in the control group dropped out. In the test group a decrease in serum total cholesterol (
6.2%, P ¼ 0:006), LDL cholesterol (
7.5%, P ¼ 0:032), triglycerol (
13.3%, P ¼ 0:016), Apo B (
5.7%, P ¼ 0:036), glucose (
5.3%, P ¼ 0:013), and homocysteine (
9.5%, P ¼ 0:00) was observed. Any of these parameters changed in the control group.

Corresponding author. Carretera de Calasancio no. 9, 14012 Co ´rdoba, Espan ˜a. Tel./fax: +34957402062.

E-mail address: [email protected] (P. Benito). 0261-5614/$ - see front matter & 2006 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2005.12.006

ARTICLE IN PRESS 582

P. Benito et al. Conclusions: Dietary supplementation with 500 cm3 of enriched milk with o-3 fatty acid, oleic acid and folic acid, reduces serum tryglicerides, total and LDL cholesterol, Apo B, glucose and homocysteine in patients with MS. This milk is well tolerated and accepted by the patients. & 2006 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Introduction The term metabolic syndrome (MS) describes a cluster of diseases with high prevalence, mainly type 2 diabetes mellitus, android obesity, hypertension and dyslipoproteinemia.1 The pathogenic basis are not clear. Two potential etiological categories have been postulated: insulin resistance with increased plasma insulin levels and central obesity, both favouring a chronic inflammatory status.2,3 Recently, new definitions of this syndrome has been propossed,4 producing a substantial confusion and a considerable doubt regarding its value as a cardiovascular risk marker.5 Nevertheless, all these factors are atherogenic, in such a way that patients with MS are at high risk of suffering an acute cardiovascular event. At present, the more useful definition is still considered to be the ATP III one, that requires three factors among the increased waist circumference, high blood pressure, hypertriglyceridemia, low cholesterol, and increased fasting glucose.6 Modification of dietary fat composition, mainly free fatty acid, can improve the lipid and carbohydrate metabolism, thus decreasing cardiovascular risk. In fact, a high consumption of fish and o-3 polyunsaturated fatty acids (PUFAs), have been associated with a lower coronary heart disease incidence and total mortality.7–9 Several mechanisms explaining this cardioprotective effect have been suggested including antiarhythmic, hypolipemic, antithrombotic and antiinflammatory properties. With this evidence, the American Heart Association recommends the intake of 1 g of o-3 per day, preferably from oily fish, in patients with documented coronary artery disease.10 In addition, a diet rich in oleic acid, has been shown to decrease LDL cholesterol and its oxidizability, as well as insulin resistance and endothelial function.11,12 Opposite effects are obtained with high intakes of saturated fatty acids (SFA). For this reason the Fat Consensus Statemens recomends the traditional Mediterranean diet, in which olive oil is the principal source of fats.13 Overall, we can assume that the reduction in total mortality after this diet, should be due to the cumulative effects of multiple dietary components, being the ratio

between monounsaturated to saturated lipids, one of the keystones.14 Recently, a semi-skimmed milk enriched with o-3 PUFAs, oleic acid, folic acid and vitamin E, has been commercialized. This milk has proved to decrease triglycerol, total cholesterol and LDL colesterol plasma levels in volunteers with or without hyperlipidemia.15,16 The present study was perfomed to assess the effects of a similar milk on cadiovascular risk factors in patients with MS.

Material and methods Study population: Patients were included, according to the ATP III concept of MS,6 with three or more of the following risk factors: waist circumference more than 102 cm in men or 88 cm in women, tryglicerol levels above 150 mg/dl, HDL cholesterol levels lower than 40 mg/dl in men and 50 mg/dl in women, blood pressure higher than 130/85 mmHg, and fasting blood glucose levels higher than 110 mg/dl. Patients with any of the following conditions were excluded: bad control of baseline illness, alcohol intake over 40 g a day, and renal or liver disease. Usual fish, meat and olive oil intake was estimated with a food-frequency questionnaire at the beginning and at the end of the study. We encouraged them not to change their physical activity and their usual diet, medication or lifestyle. Study design: The study was a controlled and open-label clinical trial, of parallel design in patients with MS. Seventy-two patients were randomly assigned to two groups: 36 patients in the test group consumed 500 cm3 per day of enriched milk (COVAPs) for 3 months, and 36 patients in the control group consumed 500 cm3 per day of semi-skimmed milk (COVAPs) for 3 months. Milk was enriched by adding o-3 (0.04 g/100 cm3) and oleic acid (1.14 g/100 cm3), and replacing the same amount of SFA, in such a way that the total fat contents of both milks were similar. Folic acid (30 mg/100 cm3) and vitamins E (1.5 mg%) were also added to this milk. The composition and the fatty acid profile of both milks are shown in Table 1.

ARTICLE IN PRESS Enriched milk effects in patients with metabolic syndrome Table 1

583

Differences in composition (per 100 ml) between the enriched and semi-skimmed milks.

Kcals Carbohydrates (g) Proteins (g) Fat (g) Oleic acid (g) Linoleic acid (g) Linolenic acid (g) Eicosopentanoic acid (g) Docosohexaenoic acid (g) Saturated fatty acids (g) Folic acid (mg) Vitamin E (mg)

Enriched milk

Semi-skimmed milk

52 5.2 3.5 1.90 1.14 0.13 0.0049 0.0030 0.0342 0.56 30 1.5

47 4.85 3.16 1.90 0.37 0.20 Undetectable Undetectable Undetectable 1.33 Undetectable Undetectable

Patients were seen every 12 days to receive the milk and to verify the compliance by collecting the empty containers. Compliance with consumption of the milk, was also confirmed by measuring fatty acids contents of serum phospholipids. At the beginning and at the end of the study a complete physical examination, including body mass index, waist circumference, blood pressure and cardiac frequency (ECG when necessary), was performed. Study was conducted according to the Helsinki Declaration. The protocol was approved by the ethical committee of our hospital and written informed consent was obtained from all subjects before inclusion. Laboratory methods: Blood samples were drawn in the hospital laboratory and were immediately processed. Serum for total and HDL cholesterol, apolipoprotein B, triglycerol, glucose, insulin, highsensitivity C-reactive protein (hs-CRP), homocysteine and fatty acids contents in serum phospholipids, was obtained at the beginning and at the end of the study. Blood glucose and lipids were also measured at 2 months. Measuring of insulin was performed by Microparticle Enzyme Immunoassay Technology (MEIA) and homocysteine by Fluorescence Polarization Immunoassay (FPIA), both in Axssym Plus analyzer (Abbotts, Chicago, IL, USA). The remaining analytical determinations were performed in ISE-2-DDPP Modular Analytics (F. Hoffman-La Roches, Basel, Switzerland) by spectrophotometric methods, except for LDL cholesterol that was calculated by the Friedewald formula. Insulin resistance was calculated by HOMA-IR ratio with the formula: fasting glucose (mmol/l)  fasting insulin (mU/l)/25. Fatty acids composition of serum phospholipids was carried out in a Hewlett Packard gas chromatograph.12

Statistical analysis: The sample size of the trial was calculated to detect a 20% reduction in serum triglycerides. It was estimated that 30 patients would be required in each group for a 2-tailed a of 0.05 and a 1
b of 80%. The drop-out rate of patients was estimated at 20%. Descriptive parameters (mean, standard deviation, absolute frequency and percentage) were estimated with 95% confidence intervals. Data were tested for normality (Kolmogorov–Smirnov statistic with Lillefors correction). Student’ t- or Mann–Whitney’s tests were applied for continuous variables. w2 (Yates corrected w2 if necessary) or exact Fisher’s tests when appropriate were used for categorical data. Significance of the change from baselines was measured by GLM Repeated Measures and the post hoc multiple comparison tests were performed for each dependent variable separately. A probability value less than 0.05 was considered to be significant. Data were analysed using SPSSs 11.0 for Windowss.

Results All but six patients (four in the test group and two in the control group) completed the study. Reasons for withdrawal were unwillingness to continue the study (two patients in each group) or moving away (two patients in the test group). Both milks were well tolerated and accepted by the patients. All of them, meat the criteria for MS according to the ATP III panel.6 Mean age (M7SD) in the test group was 52711 years, BMI 3575, waist circumference 108712 cm, systolic blood pressure 151719 mmHg, and diastolic blood pressure 92710 mmHg. Mean age (M7SD) in the control

ARTICLE IN PRESS 584

P. Benito et al.

group was 5078 years, BMI 3575, waist circumference 11079 cm, systolic blood pressure 146717 mmHg, and diastolic blood pressure 9177 mmHg. Baseline demographic and clinical characteristics of the patients that were randomized, did not differ significantly between groups. A decrease in BMI (0.6 kg/m2, P ¼ 0:001) was detected in the test group as well as in the control group (0.5 kg/m2, P ¼ 0:001). Both systolic and diastolic blood pressure fell in the test group (
9.7 mmHg, P ¼ 0:005 and
7.3 mmHg P ¼ 0:000 respectively), but only sistolic blood pressure showed a trend to decrease in the control group (
6.5%, P ¼ 0:058). At baseline, there was no differences between groups in oleic acid and o-3 fatty acids percentage in serum phospholipids. Both showed a non-significant trend to increase in the test group (+1.64% P ¼ 0:078, and +2.42% P ¼ 0:056, respectively) but not in control group. On the opposite, SFA exhibited a moderate but non-significant tendency to decrease, only in the test group (
3.82%, P ¼ 0:3). At the end of the trial, patients taking the enriched milk decreased serum total cholesterol from 223743 to 209743 mg/dl (
6.2%, P ¼ 0:006), LDL cholesterol from 133739 to 123739 mg/dl (
7.5%, P ¼ 0:032), triglycerol from 172789 to 149770 mg/dl (
13.3%, P ¼ 0:016) and Apo B from 105720 to 99721 mg/dl (
5.7%, P ¼ 0:036). Plasma glucose levels decreased significantly from 113719 to 107716 mg/dl (
5.3%, Po0.016) in this group of patients, with no change in serum insulin levels or HOMA-IR. Homocysteine decreased significantly from 11.573 to 10.473 mmol/l (
9.5%, P ¼ 0:000). CRP plasma levels remained unaltered (Table 2).

All these biochemical parameters remained unchanged in patients taking semi-skimmed milk (Table 2). The mean glucose change from baseline in the control group was obtained after 2 months of intake of the enriched milk (from 113719 to 104714 mg/ dl, Po 0.001) with a moderate rise 1 month later (107716 mg/ml, Po0.05). In contrast, the maximal decrease in plasma tryglicerol, total and LDL cholesterol levels, were obtained at the end of the study in these patients (Table 2).

Discussion A great number of cardiovascular risk factors are increased in patients with MS. These include diabetes or altered glucose metabolism as the main manifestation of insulin resistance, central obesity, high blood pressure, and increased triglycerol and LDL cholesterol with decreased HDL cholesterol. Non-classical cardiovascular risk factors like homocysteine, ultrasensible C reactive protein (CRP) or adhesion molecules such as VCAM-1 are also increased in patients with MS.1,17 Therefore, a dietary intervention capable of ameliorating these factors, is completely justified. The combined effect of o-3 and o-9 PUFAs supplementation, with an equivalent decrease in SFAs, has not been evaluated yet in patients with MS. The enriched milk is a good alternative since it was very well tolerated by the patients, and compliance was good, in fact there was no difference in the withdrawal of the study between both groups. Substitution of the usual milk for the enriched milk

Table 2 Effect of the enriched milk (test group) and semi-skimmed milk (control group) on metabolic parameters at different time points. Test group

Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglycerol (mg/dl) Apo B (mg/dl) Glucose (mg/dl) Insulin (mU/l) Homocysteine (mmol/l) CRP (mg/l) ND, not determined.  Po0.05.  Po0.01.  Po0.001.

Control group

Baseline

2 months

3 months

Baseline

2 months

3 months

223.9743 55.8715 133.6739 172.7789 105720 113719 1779.4 11.573.4 5.876

213.2740 56.5716 126.1735 152.9767 ND 104714 ND ND ND

209.9743 56.2715 123.7739 149.5770 99.8721 107.7716 16.976.9 10.473.34 4.272.6

214.1732 55.6712 128.4733 150.2768 101.8718 108.3716 15.576 11.274.9 4.3175.2

215729 55.478 129.1731 149.6764 ND 105.8714 ND ND ND

217.9727 55.9711 131.7729 151.1766 100.4717 105.3713 16.578 11.274.2 3.7673.6

ARTICLE IN PRESS Enriched milk effects in patients with metabolic syndrome increases daily intake of o-9 and o-3 PUFAs in 5.7 and 0.2 g, respectively, and decreases SFAs intake in a similar amount. Patients compliance with consumption of the milk was confirmed by the upward trend of the oleic acid and o-3 fatty acids contents of serum phospholipids in the test group. This study was performed in an inland city in the south of Spain, in which the usual diet has enough oleic acid, but less than two servings of fish per week (recommended intake of o-3 PUFA).10 Body mass index was reduced in both groups at the end of the trial, in spite of our recommendation to the patients not to change their usual diet. In fact, dietary data were collected at the beginning and at the end of the study with no significant changes. We believe that this weight reduction has not clinical relevance, and could be the result of the surveillance within a clinical trial. As weight reduction was similar in both groups, we do not think that it could justify the positive effect of the enriched milk on lipid profile and plasma glucose levels in the test group. Patients taking the enriched milk reduced systolic and diastolic blood pressure, whereas those in the control group did not show significant changes. This effect could be favoured by weight loss, however, it is more probable that the combined actions of both o-3 and oleic fatty acids could be responsible for this positive effect, since it has been reported that diet supplementation with o-3 and substitution of SFAs by monounsaturated fatty acids (MUFAs) can reduce blood pressure.10,18,19 A significant reduction of 13% in triglycerol levels (P ¼ 0:016), 6% in total cholesterol levels (P ¼ 0:006), 7.5% in LDL cholesterol (P ¼ 0:032) and 5.7% in Apo B (P ¼ 0:036) was observed in the test group but not in the control group. It has been consistently reported that substitution of SFAs by MUFAs in the diet reduces LDL cholesterol, triglycerol and Apo B levels, in normal subjects as well as in patients with hyperlipidemia or type 2 diabetes mellitus.20–22 The effect of o-3 on lipid profile is not so clear. An intake of o-3 fatty acids ranging from 3 to 4 g/d consistently decreases serum triglyceride concentrations, whereas its effects on LDL and HDL cholesterol remain controversial.10,23,24 The potential increase of LDL oxidizability caused by o-3 fatty acids, recommends the addition of an antioxidant like vitamin E to the diet of patients who are taking large amounts of fish oil.25 Postprandrial triglyceridemia reduction is especially sensitive to chronic o-3 fatty acid consumption,26 and this response is comparable in diabetic and non-diabetic subjects.27 Overall, we believe that the mentioned effect of the enriched milk on lipid profile, should be due to the

585

cumulative effects of the increase in MUFA/SFA ratio, as well as the moderate increase in o-3 intakes. High intakes of o-3 and o-9 PUFAs, folic acid and vitamin E, are important characteristics of the Mediterranean diet, so that, the enrichement of milk with these nutrients allows approaching the diet of our patients to the Mediterranean diet. Our results are in according with those of Trichopoulou, who concluded that the beneficial effects of this diet are due to a high ratio MUFA/SFA combined with a high intake in vegetables (rich in folic acid) and nuts (rich in o-3 and vitamin E).14 We confirm the results obtained by Baro et al.15 by giving a similar enriched milk (with o-3 and o-9 PUFAs, folic acid and vitamin E) to 30 healthy volunteers. After 3 months intake of the milk, they observed a significant decrease in plasma concentration of total and LDL cholesterol, and plasma homocysteine levels. Carrero et al.,16 by giving this enriched milk to 30 subjects with mild hyperlipidemia, also obtained a significant decrease of triglycerol, total and LDL cholesterol. Patients in the test group but not in the control group reduced fasting glucose levels, showing not changes in plasma insulin nor in HOMA-IR ratio. As this effect was higher in month 2 than at the end of the study, it probably has no clinical relevance and must be confirmed. The influence of o-3 fatty acid intake on glucose metabolism is not clear: Several studies have shown that fish oil has no influence on it,28 others demonstrated a glucose increase without changes in insulin secretion in type 2 diabetic patients,29 and others have shown that insulin sensitivity is significantly associated with a higher percentage of linolenic acid.30 It has been reported that there is an association between the intake of oleic acid and peripheral insulin action,12,30 and that patients with a high intake of oleic acid have a higher glucose effectiveness,11 a feature not always confirmed.31 Patients with MS and insulin resistance have a high serum CRP as a manifestation of a proinflammatory status.32,33 This is consistent with our results, since patients in both groups had a concentration of serum CRP above 3 mg/l. It has been reported that serum CRP correlates negatively with eicosapentanoic and docosahexaenoic acids,30 and that n-3 fatty acids administration have a powerful antiinflammatory property,34 although these features have not been always confirmed.35 In contrast, oleic acid does not seem to modify CRP plasma levels.36 In our study, we did not obtain any significant change in CRP in patients in the test group. Our results may indicate a lack of effect of o-3 and o-9 PUFAs on CRP, although they

ARTICLE IN PRESS 586

P. Benito et al.

can also be explained by the low amounts of these acids that were given. Hyperhomocysteinemia has been associated with insulin resistance, and could be partially responsible for increased cardiovascular risk in patients with MS.17,37 Although at baseline our patients did not show hyperhomocysteinemia, a decrease in plasma homocysteine levels at the end of the trial in the test group was detected. This effect can be attributed to the folic acid content of the enriched milk, as the effect of n-3 PUFA supplementation in the diet is under controversy.38–40 In conclusion, current diet supplementation with milk containing oleic and o-3 fatty acids plus folic acid and vitamin E, reduces several cardiovascular risk factors in patients with MS. Our results must be confirmed in more prolonged studies, taking into account final cardiovascular outcomes.

Acknowledgements This work was supported grant from the Medical Cordoba. We thank Susan manuscript and COVAP SA milk.

by a clinical research College Fundation of Webb for revising the for the free supply of

References 1. Grundy S, Brewer HB, Cleeman J, Smith S, Lenfant C for the Conference participants. Definition of metabolic syndrome. Report of the National Heart, Lung and Blood Institute/ American Heart Association Conference on Scientific Issues Related to Definition. Circulation 2004;109:433–83. 2. DeFronzo R, Ferranini E. Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipemia, and atherosclerotic cardiovascular disease. Diabetes Care 1991;14:173–94. 3. Ferna ´ndez-Real JM, Ricart W. Insulin resistance and chronic cardiovascular inflammatory syndrome. Endocrine Rev 2003;24:278–301. 4. Alberti KGM, Zimmet P, Shaw J, IDF Epidemioloy Task Force Consensus Group. The metabolic syndrome—a new worldwide definition. Lancet 2005;366:1059–62. 5. Kanh R, Ferranini E, Buse J, Stern M. The metabolic syndrome: time for a critical appraisal. Joint statement from the American Diabetes Association and the European Association for the Study of Diabetes. Diabetes Care 2005; 28:2289–304. 6. Executive Summary of the Third Report of the National Cholesterol Education Program (NECP). Expert panel on detection, evaluation and treatment of high blood cholesterol in adults (Adult Treatment Panel III). J Am Med Assoc 2001;285:2486–97. 7. Burr ML, Fehily AM, Gilbert JF, et al. Effect of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: DIET and reinfarction trial (DART). Lancet 1989;II:757–61.

8. Marchiolo R, Barzi F, Bomba E, et al. Early protection against sudden death by n-3 polyunsaturated fatty acid after myocardial infarction: time-course analysis of the results of the Gruppo Italiano per lo studio della sopravvivenza nell0 Infarto Miocardico (GISSI)-Prevenzione. Circulation 2002;105:1897–903. 9. Hu FB, Eunyoung C, Rexrode CM, Albert CM, Manson JE. Fish and long-chain o-3 fatty acid intake and risk of coronary heart disease and total mortality in diabetic women. Circulation 2003;107:1852–7. 10. Kris-Etherton PM, Harris WS, Appel LJ. AHA scientific statement: fish consumption, fish oil, omega-3 fatty acids and cardiovascular disease. Circulation 2002;106:2747–57. 11. Ryan M, McInerney D, Owens D, Collins P, Jonhson A, Tomkin GH. Diabetes and the Mediterranean diet: a beneficial effect of oleic acid on insulin sensitivity, adipocyte glucose transport and endothelium dependent vasoreactivity. Q J Med 2000;93:85–91. 12. Soriguer F, Esteva I, Rojo-Martinez G, et al. Oleic acid from cooking oils is associated with lower insulin resistance in the general population (Pizarra study). Eur J Endocrinol 2004; 150:33–9. 13. Dietary fat Consensus Statements. Am J Med 2002; 113(Suppl. 9B):5S–8S. 14. Trichopoulou A, Costacou T, Bamia C, Trichopoulos D. Adherence to a Mediterranean diet and survival in a Greek population. New Engl J Med 2003;348:2599–608. 15. Baro ´ L, Fonolla ´ J, Pen ˜a JL, et al. N-3 Fatty acids plus oleic acid and vitamin supplemented milk consumption reduces total and LDL cholesterol, homocysteine and levels of endothelial adhesion molecules in healthy humans. Clin Nutr 2003;22:175–82. 16. Carrero JJ, Baro ´ L, Fonolla ´ J, et al. Cardiovascular effects of milk enriched with o-3 polyunsaturated fatty acids, oleic acid, folic acid, and vitamins E and B6 in volunteers with mild hyperlipidemia. Nutrition 2004;20:521–7. 17. Meigs JB, Jacques PF, Selhub J, et al. Framingham offspring study. Fasting plasma homocysteine levels in the insulin resistance syndrome: the Framingham offspring study. Diabetes Care 2001;24:1403–10. 18. Grundy SM. N-3 fatty acids. Priority for post-myocardial infarction clinical trials. Circulation 2003;107:1834–6. 19. Ferrara LA, Raimondi AS, d0 Episcopo L, Guida L, Dello Russo A, Martota Tl. Olive oil and reduced need for antihypertensive medications. Arch Intern Med 2000;160:837–42. 20. Thompsen C, Rasmussen O, Lousen T, et al. Differential effects of saturated and monounsaturated fatty acids on postprandrial lipemia and incretin responses in healthy subjects. Am J Clin Nutr 1999;69:1135–43. 21. Grundy SM. What is the diserable ratio of saturated, polyunsaturated, and monounsaturated fatty acids in the diet? Am J Clin Nutr 1997;66:988S–90S. 22. Ruiz Gutierrez V, Morgado N, Prada JL, Perez-Jime ´nez F, Muriana FJ. Composition of human VLDL triacylglicerol after ingestion of olive oil and high oleic sunflower oil. J Nutr 1998;128:570–6. 23. Bucher HC, Hengstler P, Schindler C, Meier G. N-3 polyunsaturated fatty acids in coronary heart disease: a meta-analysis of randomized controlled trials. Am J Med 2002;112:298–304. 24. Goh YK, Jumpsen JA, Ryan EA, Clandinin MT. Effect of omega 3 fatty acid on plasma lipids cholesterol and lipoprotein fatty acid content in NIDDM patients. Diabetologı´a 1997;40:45–52. 25. Nestel PJ. Fish oil and cardiovascular disease: lipids and arterial function. Am J Clin Nutr 2000;71:228–31.

ARTICLE IN PRESS Enriched milk effects in patients with metabolic syndrome 26. Roche HM, Gibney MJ. Postprandrial triacylglycerolaemia: the effect of low-fat dietary treatment with and without fish oil supplementation. Eur J Clin Nutr 1996;50:617–24. 27. Montori VM, Farmer A, Wollna PC, Dinneen SF. Fish oil supplementation in type 2 diabetes: a quantitative systematic review. Diabetes Care 2000;23:1407–15. 28. Sirtori CR, Paoletti R, Mancini M, et al. N-3 fatty acids do not lead to an increased diabetic risk in patients with hyperlipidemia and abnormal glucose tolerance. Italian fish oil multicenter study. Am J Clin Nutr 1997;65:1874–81. 29. Woodman RJ, Mori TA Burke V, Puddey IB, Watts GF, Beilin LJ. Effect of purified eicosapentanoic and docosahexaenoic acid on glycemic control, blood pressure, and serum lipids in type 2 diabetic patients with treated hypertension. Am J Clin Nutr 2002;76:1007–15. 30. Fernandez Real JM, Broch M, Vendrell J, Ricart W. Insulin resistance, inflammation, and serum fatty acid composition. Diabetes Care 2003;26:1362–8. 31. Louheranta AM, Sarkkinen ES, Vidgren HM, Schwab US, Uusitupa V. Association of the fatty acid profile of serum lipids with glucose and insulin metabolism during 2 fatmodified diets in subjects wit impaired glucose tolerance. Am J Clin Nutr 2002;76:331–7. 32. Wannamethee SG, Lowe GD, Shaper AG, Rumley A, Lennon L, Whincup PH. The metabolic syndrome and insulin resistance: relationship to haemostatic and inflammatory markers in older nondiabetic men. Atherosclerosis 2005; 181:101–8. 33. Ridker P. Expert opinions: clinical application of C-reactive protein for cardiovascular disease detection and prevention. Circulation 2003;107:363–9.

587

34. Zampelas A, Panagiotakos DB, Pitsavos C, et al. Fish consumption among healthy adults is associated with decreased levels of inflammatory markers related to cardiovascular disease: the ATTICA study. J Am Coll Cardiol 2005;46:120–4. 35. Vega-Lopez S, Kaul N, Devaraj S, Cai RY, German B, Jialal I. Supplementation with omega-3 polyunsaturated fatty acids and all-rac alpha-tocopherol alone and in combination failed to exert an anti-inflammatory effect in human volunteers. Metabolism 2004;53:236–40. 36. Freese R, Vaarala O, Turpeinen AM, Mutanem M. No difference in platelet activation or inflammation markers after diets rich or poor in vegetables, berries and apple in healthy subjects. Eur J Nutr 2004;43:157–82. 37. Oron-Herman M, Rosenthal T, Sela BA. Hyperhomocysteinemia as a component of syndrome X. Metabolism 2003;52: 1491–5. 38. Grundt H, Nilsen DW, Hetland O, Mansoor MA. Clinical outcome and atherothrombogenic risk profile after prolonged wash-out following long-term treatment with high doses of n-3 PUFAs in patients with an acute myocardial infarction. Clin Nutr 2004;23:491–500. 39. Grundt H, Nilsen DW, Mansoor MA, Hetland O, Nordoy A. Reduction in homocysteine by n-3 polyunsaturated fatty acids after 1 year in a randomised double-blind study following an acute myocardial infarction: no effect on endothelial adhesion properties. Pathophysiol Haemost Thromb 2003;33:88–95. 40. Piolot A, Blache D, Boulet L, et al. Effect of fish oil on LDL oxidation and plasma homocysteine concentrations in health. J Lab Clin Med 2003;14:41–9.