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. 2022 Apr 12;14(8):1608. doi: 10.3390/nu14081608

Table 1.

Characteristics of the studies included in the systematic review.

Reference Year Location Participants Design Intervention Duration Outcomes
Haglund et al. [12] 1993 Sweden n = 12, male volunteers, healthy or with slightly to moderately increased blood lipids A double-blind cross-over study 30 mL of fish oil with 30 mL of orange juice/day for 4 weeks followed by a 5-week washout, and then 30 mL of fish oil with 30 mL of orange juice/day supplemented with vitamin B6 (80 mg/day) and folic acid (10 mg/day) for 4 weeks.
Fish oil contained 13% DHA and 19% EPA.		 Fish oil-
supplemented orange juice for 4 weeks followed by a 5-week washout period and then B vitamins + fish oil-supplemented orange juice for 4 weeks

  1. ↓Atherogenic index (12%) after fish oil alone and (24% ) after B vitamins-supplemented fish oil;

  2. ↓Plasma fibrinogen (6%) after fish oil alone and (15%) after B vitamins-supplemented fish oil;

  3. ↓TG and↑HDL-C after fish oil with and without B vitamins supplementation;

  4. ↓Plasma Hcy (30%) after B vitamins-supplemented fish oil.
Baró et al. [24] 2003 Spain n = 30, healthy volunteers (50% man) CS without control 500 mL/day of semi-skimmed milk for 4 weeks and then 500 mL/day of the enriched milk (containing omega-3 PUFAs, oleic acid, vitamins E, B6, and folic acid) for 8 further weeks. 8 weeks

  1. ↓Plasma total cholesterol (6%), LDL-C (9%), Hcy level (13%), LDL hydroperoxides (12%), and VCAM-1(16%) after intervention;

  2. No change: plasma TG and HDL-C.
Carrero et al. [26] 2004 Spain n = 30, volunteers (50% man) with mild hyperlipidemia CS without control 500 mL/day of semi-skimmed milk for 4 weeks and then 500 mL/day of the enriched milk (containing omega-3 PUFAs, oleic acid, vitamins E, B6, and folic acid) for 8 weeks. 8 weeks

  1. ↓Plasma TG (24%), total cholesterol (9%), and LDL-C (13%), VCAM-1 (9%) and Hcy (17%) after intervention;

  2. No change: Plasma MDA and LDL oxidation.
Carrero et al. [20] 2005 Spain n = 60, patients with PVD and intermittent claudication (100% man) RCT with parallel-
group design		 Supplemented group: 500 mL/day of a fortified dairy product containing EPA, DHA, oleic acid, folic acid, and vitamins B6, D, A, and E;
Control group: 500 mL/day of semi-skimmed milk with added vitamins A and D.		 12 months

  1. ↓Plasma total cholesterol and ApoB in supplement group;

  2. ↓Total Hcy in patients with high initial concentrations;

  3. ↑Walking distance before the onset of claudication and ankle-brachial pressure index values in supplement group;

  4. No change: plasma MDA, oxidized LDL, TG, CRP, PAI-1, ICAM-1, VCAM-1 and E-selectin.
Benito et al. [21] 2006 Spain n = 72, patients with metabolic syndrome A randomized, placebo-
controlled and open clinical trial of parallel design		 Control group: 500 mL semi-skimmed milk/day;
Test group: 36 patients consumed 500 mL enriched milk (5.7 g of oleic acid, 0.2 g of omega-3 PUFAs, 150 μg of folic acid and 7.5 mg of vitamin E) /day.		 3 months

  1. ↓Serum total cholesterol (6.2%), LDL-C (7.5%), TG (13.3%), Apo B (5.7%), plasma glucose (5.3%) and Hcy (9.5%);

  2. No change: serum insulin levels and plasma CRP.
Carrero et al. [23] 2006 Spain n = 40, male PVD patients A longitudinal, randomized, controlled study Group 1: 500 mL/day fortified dairy product containing fish oil, oleic acid, folic acid, vitamins A, D, E, and B6;
Group 2: 500 mL/day fortified dairy product + simvastatin (20 mg/day);
Group 3: 500 mL/day semi-skimmed milk;
Group 4: 500 mL/day semi-skimmed milk + simvastatin (20 mg/day).		 12 months

  1. Groups 1 and 2 tripled their claudication distance which correlated with ↑ plasma DHA (r = 0.40);

  2. ↑ ankle-brachial index and ABI in group 2;

  3. ↓plasma Hcy in group 1 and 2;

  4. ↑total and LDL-C in group 3.
Carrero et al. [22] 2007 Spain n = 40, patients suffered from MI (100% man) RCT with parallel-
group design		 Supplemented group: 500 mL/day of a fortified dairy product containing EPA, DHA, oleic acid, folic acid, and vitamins B6, D, A, and E;
Control group: 500 mL/day of semi-skimmed milk with added vitamins A and D.		 12 months

  1. ↓Plasma total and LDL-C, apolipoprotein B, and CRP in the supplemented group;

  2. ↓plasma total Hcy in both groups, compared to the baseline;

  3. No change: plasma HDL-C and TG, heart rate, blood pressure, or cardiac electrocardiographic parameters.
Fonollá et al. [18] 2009 Spain n = 297, participants (84.5% man) with moderate cardiovascular risk RCT with parallel-
group design		 Group 1: 500 mL/day enriched milk (containing omega-3 PUFAs, oleic acid, vitamins E, B6, and folic acid);
Group 2: 500 mL/day skimmed milk;
Group 3 (control): 500 mL/day semi-skimmed milk.		 12 months

  1. ↑ Plasma HDL-C (4%) and↓plasma TG (10%), total cholesterol (4%), and LDL-C (6%) under enriched milk consumption.

  2. No change: serum glucose, Hcy, and CRP.
Galan et al. [14] 2010 France n = 2501 patients (79.5% man) with CVD (MI, stroke and unstable angina) RCT with a 2 × 2 factorial design Group 1: B-vitamins [5-methyl-THF (560 μg), vitamin B6 (3 mg) and vitamin B12 (20 μg)] and a placebo capsule for omega-3 PUFAs;
Group 2: omega-3 PUFAs (600 mg of EPA and DHA at a ratio of 2:1) and a placebo capsule for B-vitamins;
Group 3: both B-vitamins and omega-3 PUFAs;
Group 4: placebo capsules for both treatments.		 4.7 years

  1. ↓ plasma Hcy (19%) under B vitamins treatment with no effects on major vascular events [75 vs. 82 patients; HR = 0.90, 95% CI (0.66, 1.23)];

  2. Allocation to omega-3 PUFAs had no effect on major vascular events [81 vs. 76 patients; HR = 1.08, 95% CI (0.79, 1.47)].
Szabo et al. [17] 2012 France n = 2501, patients (79.5% man) with a history of CVD (MI, stroke and unstable angina) RCT with a 2 × 2 factorial design Group 1: B-vitamins [5-methyl-THF (560 μg), vitamin B6 (3 mg) and vitamin B12 (20 μg)] and a placebo capsule for omega-3 PUFAs;
Group 2: omega-3 PUFAs (600 mg of EPA and DHA at a ratio of 2:1) and a placebo capsule for B-vitamins;
Group 3: both B-vitamins and omega-3 PUFAs;
Group 4: placebo capsules for both treatments.		 4.7 years

  1. ↓ plasma Hcy (19%) under B vitamins treatment;

  2. No effect of either omega-3 PUFAs or B-vitamins supplementation on BP;

  3. Change in BP was not associated with change in Hcy.
Earnest et al. [19] 2012 UK n = 100, participants with elevated Hcy (>8.0 umol/L) RCT with 2 × 2 factorial design Group 1: placebo;
Group 2: MVit (vit C: 200 mg; vitE: 400 IU; vit B6: 25 mg; folic acid: 400 ug; vit B12: 400 ug) + placebo;
Group 3: omega-3 PUFAs (2 g omega-3 PUFAs, 760 mg EPA, 440 mg DHA) + placebo;
Group 4: MVit + omega-3 PUFAs.		 12 weeks

  1. ↓Plasma Hcy under supplementation of MVit, OR = −1.43 [95% CI (−2.39, −0.47)] and MVit + omega-3 PUFAs, OR = −1.01, [95% CI (−1.98, −0.04)], compared to placebo and omega-3 PUFAs group;

  2. ↓Plasma CRP under supplementation of MVit, OR = −6.00 [95% CI (−1.04, −0.15)] and MVit + omega-3 PUFAs, OR = −0.98 [95% CI (−1.51, −0.46), but not vs. placebo or omega-3 PUFAs group;

  3. ↓Plasma TG under supplementation of omega-3 PUFAs, OR = −0.41 [95% CI (−0.69, −0.13)] and MVit + omega-3 PUFAs, OR = −0.71 [95% CI (−0.93, −0.46)], compared to placebo and omega-3 PUFAs group.
De Natale C et al. [4] 2012 Italy n = 16, participants (43.8% man) with mild plasma lipid abnormalities a randomized crossover design Group 1: a diet containing baked products enriched with active nutrients [β-glucans (3.6 g/day), folic acid (1620 μg/day), long-chain (800 mg/day) and short-chain (400 mg/day) omega-3 PUFAs, and tocopherols (120 mg/day);
Group 2: a diet containing the same products without active nutrients (control diet).		 1 month for each of the control and enriched diets and then cross over to the other diet

  1. ↓Fasting plasma TG after the active vs. control diet (1.56 ± 0.18 vs. 1.74 ± 0.16 mmol/L), as was the postprandial level of chylomicron TG and the insulin peak;

  2. ↓fasting Hcy (8 ± 0.6 vs. 10 ± 0.8 μmol/L) and hunger feeling at the fifth and sixth hour after active diet;

  3. No change: CRP.
Blacher et al. [6] 2013 France n = 2501, patients (79.5% man) with a past history of cardio- or cerebrovascular diseases RCT with a 2 × 2 factorial design Group 1: B-vitamins [(5-methyl-THF (560 μg), vitamin B6 (3 mg) and vitamin B12 (20 μg)] and a placebo capsule for omega-3 PUFAs;
Group 2: omega-3 PUFAs (600 mg of EPA and DHA at a ratio of 2:1) and a placebo capsule for B-vitamins;
Group 3: both B-vitamins and omega-3 PUFAs;
Group 4: placebo capsules for both treatments.		 4.2 (± 1.0) years

  1. Neither omega-3 PUFAs treatment, nor B vitamins treatment was associated with the occurrence of hard coronary events;

  2. Allocation to omega-3 PUFAs was not associated with any significant effect on coronary revascularization;

  3. B vitamins treatment was associated with 52% increase in the risk of coronary revascularization [HR = 1.52, 95% CI (1.11–2.10)].
Garaiova et al. [5] 2013 Slovakia n = 25, hypercholesterolmic children and adolescents, mean age 16.4 ± 3.8 years CS without control A combination of plant sterols esters (1300 mg), fish oil (1000 mg EPA plus DHA) and vitamins B12 (50 μg), B6 (2.5 mg), folic acid (800 μg) and coenzyme Q10 (3 mg). 16 weeks

  1. ↓serum total cholesterol, LDL-C, VLDL-C, subfractions LDL-2, IDL-1, IDL-2 and plasma Hcy;

  2. ↓α-tocopherol levels after standardisation for LDL-C;

  3. ↑Omega-3 PUFAs with the dietary supplementation;

  4. No change: TG, CRP, HDL-cholesterol and apolipo-protein A1.
Huang et al. [3] 2015 China n = 38, healthy individuals, 57% man, 23 ± 3 years of old RCT with parallel-
group design		 Group 1: vitamin B12 (1000 μg);
Group 2: fish oil (2 g);
Group 3: vitamin B12 (1000 μg) + fish oil (2 g) (each 1 g capsule provided 490 mg of 22:6 omega-3, and 98 mg of 20:5 omega-3).		 8 weeks

  1. ↓Plasma TG, uric acid, CRP, and ferritin after 4 and 8 week supplementation of fish oil, and vitamin B12 + fish oil;

  2. ↓plasma Hcy by 22%, 19%, and 39% after vitamin B12, fish oil, and vitamin B12 + fish oil supplementation respectively.

PUFAs, polyunsaturated fatty acids; CVD, cardiovascular diseases; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; Hcy, homocysteine; CS, cross-sectional study; LDL-C, low-density lipoprotein cholesterol; VCAM-1, vascular cell adhesion molecule-1; MDA, malondialdehyde; PVD, peripheral vascular disease; RCT, randomized controlled trial; ApoB, apolipoprotein B; CRP, C-reaction protein; PAI-1, plasminogen activator inhibitor-1; ICAM-1, intercellular adhesion molecule-1; ABI, ankle-brachial pressure index; MI, myocardial infarction; THF, tetrahydrofolic acid; HR, hazard ratio; CI, confidence interval; BP, blood pressure; MVit, multivitamins; OR, odds ratio; VLDL-C, very low-density lipoprotein cholesterol; ↓, decrease; ↑, increase.