sign in sign up
<<
Home , Folate

Food Suppl Biomater Health. 2021 Jun;1(2):e24 https://doi.org/10.52361/fsbh.2021.1.e24 pISSN 2765-4362·eISSN 2765-4699

Original Article Genetic Variation in MCP-1 Is Associated with the Efficacy of Spirulina Supplementation on Antioxidant Levels in Elderly Korean: a Randomized Double Blind Trial

Hye Ran Lee ,1 Hee Jung Park 2

1Department of Food & , Baewha Women's University, Seoul, Korea 2Department of Food & Nutrition, Sangmyung University, Seoul, Korea

Received: Jun 6, 2021 Revised: Jun 24, 2021 ABSTRACT Accepted: Jun 24, 2021 Spirulina is a known a functional food related to antioxidant capacity. Gene variation may Correspondence: impact functional food supplementary effects. The current study investigated the interaction Hee Jung Park, PhD of monocyte chemoattractant protein-1 (MCP-1)-2518 polymorphism with spirulina Department of Food & Nutrition, Sangmyung University, 20 Hongjimun 2-gil, Jongno-gu, supplements on antioxidant capacity in Korean elderly (n = 78). The subjects consumed 8 Seoul 03016, Korea. g/day either spirulina or placebo for 4 months by using a randomized, double blind, and E-mail: [email protected] placebo-controlled design. Antioxidant level, body composition, and dietary intake were measured twice (baseline vs. week 16). Plasma antioxidant status (TAS), thiobarbituric © 2021 Health Supplements Future Forum This is an Open Access article distributed acid reactive substance (TBARS), A, , , and were analyzed as under the terms of the Creative Commons antioxidant markers. For the subject with A allele, the plasma level of antioxidant such Attribution Non-Commercial License (https:// as , and folate was significantly higher in spirulina group than placebo. The plasma creativecommons.org/licenses/by-nc/4.0/) TBARS level was significantly lower in spirulina group than placebo. For the subject with which permits unrestricted non-commercial G/G genotype, among the antioxidant , only folate level was significantly higher in use, distribution, and reproduction in any spirulina group than placebo. The plasma TAS level was significantly higher in spirulina group medium, provided the original work is properly cited. with G/G genotype. The effect of treatment × time was only found in plasma level of folate and TBARS in A/A genotype. In conclusion, MCP-1 genotype is associated with antioxidant efficacy ORCID iDs in response to spirulina supplementation. These results may be useful for personalized Hye Ran Lee nutritional recommendation to improve antioxidant capacity in healthy Korean elderly. https://orcid.org/0000-0002-0876-8730 Hee Jung Park Keywords: Genetic variation; Monocyte chemoattractant protein 1; Spirulina; Antioxidants; https://orcid.org/0000-0001-6278-7812 TBARS Funding Financial aid from the National Research Foundation of Korea (No. R04-2003-000- 10119-0) and Earth Spirulina group is gratefully INTRODUCTION acknowledged. Monocyte chemoattractant protein-1 (MCP-1) involved in cardiovascular disease.1-6 MCP- Disclosure 1 promotes the transmigration and emigration of circulating monocytes into tissues, The authors have no potential conflicts of interest to disclose. and exerts various effects on monocytes, including the induction of superoxide anions and expression of various proinflammatory genes.1,2 MCP-1 is expressed at high levels in Author Contributions atherosclerotic plaques3 and the level of MCP-1 has also been shown to increase in patients Conceptualization: Park HJ; Data curation: Lee with myocardial infarction, unstable angina, and venous thrombosis, and is considered to HR; Investigation: Park HJ; Methodology: Park 4-6 HJ; Supervision: Park HJ; Validation: Lee HR; involve in the development and progression of cardiovascular disease. https://e-fsbh.org 1/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

Visualization: Lee HR; Writing - original draft: Oxidative damage and antioxidant protection are also associated with cardiovascular Lee HR; Writing - review & editing: Park HJ. disease.7 Under oxidative stress, oxidative modifications of low- lipoprotein (LDL) and lipid peroxidation take place. And also, there is endothelial dysfunction, monocyte adhesion, macrophage uptake of lipoprotein and inflammatory process.8 Reactive oxygen species are known to stimulate the production of inflammatory mediators like interleukin (IL)-8 and MCP-1.9 Therefore, antioxidant food can ameliorate the inflammatory process. Aronia melanocarpa which constitute important phytochemicals including proanthocyanidins, anthocyanins, and hydroxycinnamic acids reduces the MCP-1 level10 and indicated inhibition of neutrophil infiltration in the injured site.11 Ko and Kim12 reported diet composition and long-term exercise training for 8 weeks decreased inflammation via antioxidant lowering effect. In the animal experiment, it was confirmed that the MCP-1 level decreased when the antioxidant gene such as hypoxia inducible factor-1 and extra cellular signal regulate kinase was suppressed.13

Single polymorphism (SNP) in the MCP-1 promoter region have been identified and shown to affect transcription of the gene. The MCP-1 gene A-2518G polymorphism confers an increased risk of vascular complications in type 2 mellitus patients.14 The 2518A/G polymorphism in the MCP-1 gene is associated with , risk, and mood disorder.15-17 Genetic variation could modulate the efficacy of functional food supplementation. Mohseni18 reported receptor gene polymorphism related the

efficacy of vitamin D3 supplementation on inflammatory marker and total antioxidant capacity among breast cancer women. Borel and Desmarchelier19 described that genetic variation is associated with postprandial vitamin A concentration and vitamin A . The effect of vitamin E supplementation on blood haptoglobin was different by Haptoglobin genotype in Singapore diabetes pateints.20 For MCP-1 gene variation, few papers have studied the effect of dietary supplements according to the MCP-1 gene polymorphism. In our previous study,21 the differences in cytokine and MCP-1 according to the MCP-1 gene polymorphism was drawn after spirulina supplementation.

Spirulina provides an adequate amount of a spectrum of carotenoids pigments, especially β-carotene and zeaxanthin which components are well known to have antioxidant properties. Spirulina is a microscopic and filamentous cyanobacterium (blue-green alga) that has a long history of use as food which is a rich source of , especially vitamin 22 B12 and provitamin A (β-carotene), minerals, carotenoids, zeaxanthin, phycocyanins. All components of spirulina are well known to have antioxidant properties. Many studies have reported that spirulina is overwhelming in the areas of cholesterol-regulatory properties, immunomodulation, antioxidant capacity, and anti-cancer.23-28 In human study, spirulina maxima decrease endothelial damage and oxidative stress indicators in patients with systemic arterial hypertension.29 And spirulina supplementation considerably improved total antioxidant status (TAS) compared to placebo-treated individuals.30 However, there are few studies on the interaction of spirulina with genetic variants.

MCP-1 is significantly increased under conditions of oxidative stress, which leads to cardiovascular disease. The supplement that can inhibit oxidative damage could decrease the expression of MCP-1, and it may interfere with cardiovascular disease progression. From here, it is important to consider that MCP-1 expression is affected by the MCP-1 genotype. Therefore, this study was performed to examine the responsiveness of food intervention according to MCP-1 genotype considering the limited evidence available on the relationships between the individual response to spirulina induced antioxidant effect and MCP-1 gene variation. https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 2/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

METHODS

Subjects and experimental design The healthy subjects for intervention study were recruited through an advertisement in local newspaper (2005.12–2006.6). The volunteers were first interviewed by telephone for screening. The exclusion criteria for subject was current user of vitamin supplements, current drug-user of inflammatory disease (e.g., Crohn disease, rheumatoid arthritis), dyslipidaemia, and hypertension, concurrent or recent participant in other intervention study. Finally, 78 subjects (male 43, female 35) were enrolled in this study. The present study protocol was reviewed and approved by the Institutional Review Board of Ewha Women's University (ECT 109-02-01). Informed consent was submitted by all subjects when they enrolled.

This study was a 16-week randomized, double-blind, placebo-controlled trial of spirulina supplementation in elderly Korean. The subjects consumed either the spirulina or placebo, 8 g in a day. During the intervention period, subjects were asked to keep usual diet, and prohibited to take any functional food or supplements. Both spirulina (100% spirulina) and placebo (100% starch) were provided by Earth Spirulina group (Seoul, Korea). Compliance was checked by calling the subjects twice a week.

Dietary intake and anthropometric measures The food consumption was assessed using 24-hour recall method. Dietary intake data was analyzed by using CAN-Pro 4.0 software (The Korean Nutrition Society, Seoul, Korea). Height was measured using an anthropometer (Seca 213; Seca Inc., Birmingham, UK). Body weight and composition were measured using INBODY 2.0 (Biospace Co., Seoul, Korea) with subjects wearing light clothing.

Blood collecting and measurement Blood samples were taken on baseline and 16th week of the study period. Serum and plasma samples were separated by centrifuge with 2,000 rpm for 25 minutes and the samples were stored at −80°C freezer until measurement. Total cholesterol and triglyceride level were measured by using autoanalyzer (Ekachem DTSC module; Johnson & Johnson, New Brunswick, NJ, USA). High-density lipoprotein cholesterol level was determined by autoanalyzer after treating UC infranatant with phosphotungstic acid-Mg. LDL-cholesterol (LDL-C) was calculated by Friedewald method.31 Plasma MCP-1 concentrations were determined by linked immunosorbent assay (ELISA) technique (Quantikine; R&D Systems Inc., Minneapolis, MN, USA) reading ELISA reader (Spectra Max 340; Molecular Devices, San Jose, CA, USA).

Plasma level of vitamin A, C, E, and folate Plasma level of and α- were determined using reversed-phase high performance liquid chromatography (HPLC). Briefly, the methanol was added to precipitate protein, and n-haxane was added to extract lipids. The mixed solution was centrifuged at 3,000 rpm for 15 minutes at room temperature. After centrifuging, the hexane layer was

moved in brown vial, and volatilized by N2 gas using a vacuum evaporator. The remainder was dissolved in 1 mL of eluent (dichloromethane:methanol = 15:85) and filtered using a 0.45 µm filter (NYLON 66 SYRINGE FILTER; Whatman, Florham Park, NJ, USA). Alpha-tocopherol and retinol were measured using HPLC (Waters 2690 Separation Module; Waters, Milford, MA, USA) equipped with UV detector (YoungLin Co. Ltd., Anyang, Korea) using a XTerra™ RP18 column (5 µm; 4.6 × 150 mm; Waters). The absorbance of samples was quantified using https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 3/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

the Autochro -WIN program (version 2.0 plus; YoungLin Instruments Co., Anyang, Korea). All extraction procedures were performed under dark conditions. Mobile phase consisted of methanol and dichloromethane (85:15, v/v). The flow rate was 0.8 mL/min. The detection wavelengths were set to 295 nm for Alpha-tocopherol and to 325 nm for retinol.

Plasma vitamin C was determined by colorimetric vitamin C assay kit (Immundiagnostik AG, Bensheim, Germany). Serum folate level was measured using 125I-folic acid and 57Co- dualcount SPNB (solid phase no boil) radioassay kit (Diagnostic Products Co., Los Angeles, CA, USA). The final concentration was determined by dualγ-counter (CobraII, auto-gamma; Mirion Technologies, Inc., San Ramon, CA, USA).

Assay of antioxidant capacity Plasma concentration of thiobarbituric acid reactive substance (TBARS) concentration was determined using Yagi method32 and TBARS concentration was determined with luminescence spectrometer (LS50; Perkin Elmer, Waltham, MA, USA) at excitation 515nm, emission 553 nm. A standard curve was constituted from serial dilutions (0–1.0 nM) of a 1,1,3,3-tetra-methoxypropane [Malonaldehyde bis (dimethyl-acetal)] standard solution. TAS of plasma sample was measured using a commercial TAS kit (Randox Laboratories Ltd, Crumlin, UK).

DNA extraction and analysis of the MCP-1 polymorphism All subjects were genotyped for MCP-1 polymorphism (dbSNP: rs1024611). The DNA was extracted from whole blood (QIAamp DNA Blood Kit; Qiagen, Hilden, Germany), and a 649 bp segment of MCP-1 gene including polymorphism sites was amplified by the polymerase chain reaction (forward primer: 5′-AGT CCA ACC AAG GTT TGT GC-3′ and the reverse primer 5′-TCA TGC TTC GGG TTT TCT CT-3′). The presence of MCP-1 polymorphism was determined by restriction fragment length polymorphism assay using PvuII enzyme.

Statistical analysis Analysis was conducted using SPSS Statistics version 24. Values are shown as the mean and standard error. The results were presented for placebo and spirulina group by genotype. Paired t-test was used to analyze mean differences for all measured parameters between pretest and posttest. To examine the effect of treatment, repeated measured analysis of variance test was conducted.

RESULTS

MCP-1 genotype and self-reported health behavior Seventy-eight healthy volunteers were enrolled in this intervention study and were divided into placebo (n = 37) and spirulina groups (n = 41) by double-blind and randomization. The subjects' age was 65.9 years, there were no significant differences in age, sex, and genotype distribution between 2 groups within same genotypes (Table 1). All participants were in good health based on the medical history and medication. More than 90% of the subjects were non- smokers, and 60% of subjects were regular exercisers. The health status and health behavior were not significantly different in both groups within same genotype and among the genotype.

https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 4/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

Table 1. Monocyte chemoattractant protein-1 genotype and self-reported health behavior of the volunteers Variables All A/A G/A G/G Placebo Spirulina Placebo Spirulina Placebo Spirulina Placebo Spirulina Number 37 (47.4) 41 (52.6) 5 (13.5) 9 (21.9) 19 (51.4) 25 (61.0) 13 (35.1) 7 (17.1) Agea 66.0 ± 1.0 65.8 ± 0.9 64.5 ± 1.7 65.9 ± 1.4 64.9 ± 1.1 64.7 ± 0.9 68.2 ± 2.0 70.1 ± 3.6 Sexb (male/female) 19/18 24/17 3/2 7/2 10/9 12/13 6/7 5/2 Subjects with diseaseb No 28 (75.7) 31 (75.6) 4 (80.0) 7 (77.7) 15 (78.9) 18 (72.0) 9 (69.2) 6 (85.7) Yes 9 (24.3) 10 (24.4) 1 (20.0) 2 (22.2 4 (21.1) 7 (28.0) 4 (30.8) 1 (14.3) Medication userb No 35 (97.6) 38 (92.7) 4 (80.0) 8 (90.0) 18 (94.7) 23 (92.0) 13 (100.0) 7 (100.0) Yes 2 (2.4) 3 (7.3) 1 (20.0) 1 (10.0) 1 (5.3) 2 (8.0) 0 (0) 0 (0) Cigarette smokerb Non-smoker 33 (89.2) 28 (68.3) 4 (80.0) 5 (55.6) 16 (84.2) 18 (72.0) 13 (100.0) 5 (71.4) Current smoker 2 (5.4) 3 (7.3) 1 (20.0) 1 (11.1) 1 (5.3) 2 (8.0) 0 (0) 0 (0) Ex-smoker 2 (5.4) 10 (24.4) 0 (0) 3 (33.3) 2 (10.5) 5 (20.0) 0 (0) 2 (28.6) Alcohol drinkerb No 22 (59.5) 21 (51.2) 3 (60.0) 4 (44.4) 10 (52.6) 15 (60.0) 9 (69.2) 2 (28.6) Yes 15 (40.5) 20 (48.8) 2 (40.0) 5 (55.6) 9 (47.4) 10 (40.0) 4 (30.8) 5 (71.4) Regular exerciserb No 14 (37.8) 16 (39.0) 2 (40.0) 2 (22.2) 7 (36.8) 11 (44.0) 5 (38.5) 3 (42.9) Yes 23 (62.2) 25 (61.0) 3 (60.0) 7 (77.8) 12 (63.2) 14 (56.0) 8 (61.5) 4 (57.1) Family history of cardiovascular diseaseb No 22 (59.5) 26 (63.4) 3 (60.0) 7 (77.8) 12 (63.2) 14 (56.0) 7 (53.8) 5 (71.4) Yes 15 (40.5) 15 (36.6) 2 (40.0) 2 (22.2) 7 (36.8) 11 (44.0) 6 (46.2) 2 (28.6) Data are shown as mean ± standard error or number (%). aNot significantly different by Student t-test between placebo and spirulina groups within same genotype (P < 0.05); bNot significantly different byχ 2 test between placebo and spirulina groups within same genotype (P < 0.05).

Baseline characteristics Baseline characteristics by supplementation group were shown in Table 2. The anthropometric parameters were not different between 2 groups within same genotype. No differences were observed in the MCP-1 level, lipid profiles, and blood pressure by the groups.

Table 2. Baseline characteristics Characteristics All A/A G/A G/G Placebo Spirulina Placebo Spirulina Placebo Spirulina Placebo Spirulina Weight (kg) 61.8 ± 1.44 64.3 ± 1.87 65.3 ± 4.65 69.0 ± 4.25 61.6 ± 1.96 64.2 ± 2.53 61.1 ± 2.54 58.3 ± 1.41 BMI (kg/m2) 24.3 ± 0.41 24.6 ± 0.49 26.3 ± 2.15 25.3 ± 1.21 24.2 ± 0.56 24.9 ± 0.62 23.9 ± 0.48 22.6 ± 0.48 Waist (cm) 85.1 ± 1.27 86.9 ± 1.57 88.7 ± 5.60 90.6 ± 3.70 84.7 ± 1.83 86.2 ± 2.13 84.5 ± 1.68 84.0 ± 1.49 WHR (cm) 0.86 ± 0.01 0.87 ± 0.01 0.86 ± 0.03 0.89 ± 0.01 0.87 ± 0.01 0.86 ± 0.01 0.86 ± 0.01 0.87 ± 0.01 TSF (mm) 23.7 ± 1.47 25.9 ± 1.42 26.0 ± 7.26 24.8 ± 3.57 23.6 ± 2.08 26.3 ± 1.78 23.2 ± 1.94 26.1 ± 3.14 Bodyfat (%) 29.2 ± 0.91 28.4 ± 0.94 31.4 ± 3.59 25.5 ± 1.07 28.3 ± 1.35 30.3 ± 1.21 29.9 ± 1.18 25.7 ± 2.59 Fasting blood (mg/dL) 100.9 ± 2.9 105.1 ± 2.1 105.8 ± 15.5 97.0 ± 4.3 98.1 ± 3.1 108.8 ± 2.8 104.0 ± 5.6 104.1 ± 3.8 Total cholesterol (mg/dL) 196.4 ± 6.2 189.4 ± 5.79 186.5 ± 27.6 181.6 ± 9.4 195.8 ± 7.9 193.6 ± 8.2 200.7 ± 10.9 185.8 ± 14.4 LDL-C (mg/dL) 126.3 ± 6.9 116.5 ± 5.83 115.1 ± 20.8 116.3 ± 9.7 124.6 ± 9.2 117.6 ± 8.5 132.9 ± 13.0 113.1 ± 13.1 HDL-C (mg/dL) 49.3 ± 2.77 48.9 ± 2.48 45.0 ± 5.2 43.9 ± 3.3 50.8 ± 4.4 48.4 ± 3.3 48.3 ± 3.77 58.3 ± 7.1 Triglyceride (mg/dL) 103.6 ± 8.7 119.5 ± 12.8 132.3 ± 45.2 107.2 ± 17.1 101.9 ± 11.2 138.4 ± 19.7 97.0 ± 12.5 72.3 ± 7.43 LDL/HDL ratio 2.84 ± 0.19 2.58 ± 0.14 2.58 ± 0.40 2.75 ± 0.28 2.79 ± 0.24 2.65 ± 0.22 3.03 ± 0.41 2.08 ± 0.31 SBP (mmHg) 136.4 ± 2.81 135.6 ± 2.38 143.1 ± 12.7 141.8 ± 4.02 131.9 ± 3.6 133.2 ± 3.2 141.1 ± 4.2 135.3 ± 6.1 DBP (mmHg) 84.9 ± 1.37 83.2 ± 1.57 89.3 ± 3.7 87.4 ± 3.7 83.4 ± 1.9 81.4 ± 2.1 86.1 ± 2.3 82.2 ± 2.2 Energy (kcal) 1,593.0 ± 89.3 1,503.9 ± 55.6 1,737.6 ± 121.2 1,662.7 ± 148.4 1,549.4 ± 122.4 1,467.6 ± 56.5 1,492.6 ± 122.2 1,401.5 ± 151.9 Vitamin A intake (µg RE) 447.0 ± 43.5 384.4 ± 29.9 381.5 ± 120.8 341.1 ± 42.7 473.3 ± 62.3 385.3 ± 46.5 349.1 ± 58.3 414.8 ± 64.8 Vitamin C intake (mg) 123.1 ± 17.5 113.2 ± 11.3 99.6 ± 51.9 127.3 ± 22.1 146.2 ± 29.3 108.1 ± 14.0 94.7 ± 12.8 110.3 ± 36.8 Vitamin E intake (mg TE) 8.77 ± 0.92 7.52 ± 0.79 9.45 ± 2.8 7.42 ± 1.5 7.56 ± 1.01 8.2 ± 1.0 9.3 ± 1.7 6.94 ± 1.2 Folate intake (µg DFE) 246.6 ± 24.2 222.9 ± 18.8 285.4 ± 43.7 236.9 ± 37.6 241.9 ± 28.5 233.0 ± 27.4 212.6 ± 19.4 198.8 ± 13.2 Data are shown as mean ± standard error or number (%). BMI = body mass index, WHR = waist-to-hip ratio, TSF = triceps skinfolds thickness, LDL-C = low-density lipoprotein cholesterol, HDL-C = high-density lipoprotein cholesterol, SBP = systolic blood pressure, DBP = diastolic blood pressure. https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 5/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

Dietary intakes were not different according to supplement group. The intake of total energy, vitamin A, C, E, and folate related to antioxidant status were similar between the groups.

Dietary intakes The subject's dietary intake during the intervention period may confuse the effect of the test substance. Therefore, by examining the amount of dietary intake before and after the supplementation, it was attempted to exclude confounding factors from supplement efficacy.

The total food intake was not changed in the subjects with A/A or G/G genotype during intervention period. There was no difference in daily nutrients intake in the subjects with A/A or G/G genotype after supplementation either spirulina or placebo (Table 3). In the G/A

genotype, daily intakes of energy, Ca, vitamin A, carbohydrate, phosphate, and vitamin B2 were changed after intervention in both groups. However, significant time-by-treatment interaction was not observed.

Plasma level of vitamin A, C, E, and folate The response for antioxidant nutrients was shown in Table 4. In all subjects, plasma level of retinol was decreased, and folate level was increased after intervention period for both groups. There were no significant differences in the plasma level of retinol and α-tocopherol level during intervention period in each genotype. In the A/A genotype, the plasma level of vitamin C and folate were increased after spirulina supplement, from 9.97 pg/mL to 11.5 pg/ mL, and from 7.63 nmol/L to 17.6 nmol/L, respectively. In the G/A and G/G genotype, the folate level was significantly increased. The effect of treatment was found in plasma level of folate (time × treatment interaction P < 0.05) in all subject and in A/A genotype group.

Antioxidants capacity The response for antioxidant status was shown in Table 5. In all subjects, TBARS level was decreased and TAS level was significantly increased after spirulina supplementationP ( < 0.05). However, no significance was found in the effect of treatment × time. In the A/A

Table 3. Background intake during intervention according to monocyte chemoattractant protein-1 genotype Nutrients All A/A G/A G/G Energy (kcal) 0.5143a 0.1806 0.5379 0.1297 Protein (g) 0.6178 0.5189 0.2122 0.0537 Fat (g) 0.9618 0.5037 0.7228 0.1615 Carbohydrate (g) 0.2917 0.7179 0.2853 0.4801 Fiber (g) 0.3075 0.1774 0.6721 0.7208 Ca (mg) 0.8989 0.6321 0.9357 0.2901 P (mg) 0.5857 0.3122 0.2708 0.1547 Fe (mg) 0.1428 0.5213 0.3169 0.1076 Zn (mg) 0.3514 0.7477 0.2364 0.1820 Vitamin A(µg RE) 0.2707 0.3547 0.2284 0.4980

Vitamin B1 (mg) 0.9320 0.3719 0.6827 0.3526

Vitamin B2 (mg) 0.7013 0.2970 0.2213 0.1252

Vitamin B6 (mg) 0.2165 0.1773 0.2417 0.6591 Vitamin C (mg) 0.8290 0.9972 0.9227 0.2794 Vitamin E (mg TE) 0.4986 0.1837 0.4183 0.7231 (mg) 0.3379 0.8380 0.1587 0.4626 Folic acid (µg DFE) 0.4613 0.3115 0.7897 0.5197 % Carbohydrateb 0.7476 0.6743 0.3138 0.0617 % Protein 0.7690 0.6360 0.1677 0.0476 % Fat 0.5214 0.9929 0.5426 0.3074 aPr > F: repeated measure analysis of variance; bThe percentage contribution of the 3 macronutrients (carbohydrate, protein, and fat) to the total daily energy intake. https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 6/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

Table 4. Antioxidant nutrient level Blood nutrient All A/A G/A G/G level Placebo Spirulina P valueb Placebo Spirulina P value Placebo Spirulina P value Placebo Spirulina P value Retinol (µg/dL) 0.8876 0.6220 0.7221 0.7883 Baseline 34.2 ± 0.90 35.6 ± 1.20 31.2 ± 0.45 37.9 ± 3.52 34.8 ± 1.25 35.3 ± 1.30 34.8 ± 1.82 33.3 ± 1.68 16th week 30.6 ± 0.15**a 30.9 ± 0.23** 30.2 ± 0.04 31.8 ± 0.69 30.7 ± 0.24 30.5 ± 0.17 30.6 ± 0.15 31.1 ± 0.51 Vitamin C (µg/dL) 0.5106 0.5583 0.3883 0.6076 Baseline 10.4 ± 0.29 10.1 ± 0.26 11.2 ± 1.26 9.97 ± 0.80** 10.2 ± 0.44 10.1 ± 0.24 10.4 ± 0.36 10.5 ± 0.70 16th week 11.6 ± 0.81 12.7 ± 1.30 11.0 ± 3.82 11.5 ± 0.63 11.5 ± 1.22 13.7 ± 2.20 12.0 ± 0.97 10.9 ± 0.33 α-tocopherol (mg/L) 0.1966 0.4836 0.3019 0.2837 Baseline 780.7 ± 11.4 826.9 ± 24.5 779.4 ± 27.9 831.1 ± 10.2 781.8 ± 2.13 822.8 ± 13.7 779.3 ± 0.27 853.8 ± 27.1 16th week 798.7 ± 13.4 779.9 ± 0.79 892.6 ± 13.9 778.9 ± 8.9 795.8 ± 16.9 780.8 ± 1.35 779.9 ± 0.59 778.6 ± 0.25 Folate (nmol/L) 0.0252** 0.0129** 0.4718 0.3258 Baseline 11.5 ± 1.25 8.70 ± 0.79 11.19 ± 2.31 7.63 ± 1.37 9.97 ± 1.18 9.63 ± 1.09 13.9 ± 2.97 7.06 ± 1.43 16th week 15.9 ± 1.56** 18.6 ± 1.46*** 10.2 ± 2.03 17.6 ± 2.62** 16.8 ± 2.40** 19.8 ± 2.17** 16.4 ± 2.30 15.8 ± 2.02** Data are shown as mean ± standard error. aSignificantly different by paired t-test between baseline and 16th week** ( P < 0.05, ***P < 0.001); bP value for interaction test between time and treatment by repeated measure analysis of variance (**P < 0.05).

Table 5. Antioxidant status based on monocyte chemoattractant protein-1 genotype Antioxidant status All A/A G/A G/G Placebo Spirulina P valueb Placebo Spirulina P value Placebo Spirulina P value Placebo Spirulina P value TAS (nmol/L plasma) 0.0897 0.2958 0.2791 0.2342 Baseline 1.64 ± 0.08 1.75 ± 0.09 1.41 ± 0.16 1.90 ± 0.15 1.74 ± 0.08 1.76 ± 0.13 1.58 ± 0.19 1.60 ± 0.20 16th week 2.12 ± 0.14**a 2.11 ± 0.13** 2.26 ± 0.32 2.11 ± 0.42 2.16 ± 0.20 2.07 ± 0.15 2.03 ± 0.25 2.25 ± 0.14** TBARS (nmol/mL plasma) 0.2065 0.0183** 0.4457 0.1261 Baseline 6.19 ± 0.72 6.91 ± 0.25 6.27 ± 0.58 7.03 ± 0.46 6.20 ± 0.26 6.52 ± 0.39 6.11 ± 0.37 7.18 ± 0.97 16th week 5.96 ± 0.23 5.69 ± 0.37** 7.21 ± 0.62** 5.27 ± 0.98 5.83 ± 0.32 6.14 ± 0.50 5.94 ± 0.39 5.54 ± 0.85 Data are shown as mean ± standard error. TAS = total antioxidant status, TBARS = thiobarbituric acid reactive substance. aSignificantly different by paired t-test between protest and posttest,** P < 0.05); bP value for interaction test between time and treatment by repeated measure analysis of variance (**P < 0.05).

genotype, the TBARS level was significantly increased in placebo group and decreased in spirulina group, from 6.27 nmol/L to 7.21 nmol/L for placebo, from 7.03 nmol/L to 5.27 nmol/L for spirulina. In the G/A genotype, TBARS and TAS level was not changed after supplementation with spirulina. In the subject with G/G genotype, TAS level was significantly increased in spirulina group, from 1.60 nmol/L to 2.25 nmol/L. The TBARS level tended to decrease in spirulina group. The effect of treatment was found in plasma level of TBARS (time × treatment interaction P < 0.05) in subject with A/A genotype.

DISCUSSION

Our study indicated that genotype-diet interaction was observed for MCP-1 variation in the response of spirulina supplement on the antioxidant capacity. Spirulina is reported to be effective to improve blood lipid profiles, to enhance immune capacity, and to reduce oxidative stress.23-28 For the antioxidant properties of spirulina, Benedetti et al.33 reported that spirulina inhibited lipid peroxidation more significantly (65% inhibition) than the other antioxidants like α-tocopherol (35%), butylated hydroxytoluene (45%), and β-carotene (48%) in vitro. In animal study,34 plasma antioxidant capacity was higher in spirulina treatment group than control group.

In terms of MCP-1 polymorphism, many data from studies have shown that A/A genotype was wild-type and G/G homozygotes frequency was increased among cardiovascular disease’s https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 7/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

patients in Europe and America.35,36 A few studies conducted with Korean population reported that the G/G genotype, not A/A genotype, was wild-type genotype.37-39 Kim et al.37 informed that the frequency of A/A, G/A, and G/G genotype among healthy Korean was 20%, 42%, 38%, respectively, and Pae et al.38 also found that A/A genotype was 9.6% in healthy Korean population. Hwang et al.39 reported that A/A genotype and G/G genotype were 16% and 46%, respectively, in the healthy Koreans. The frequency of the G-allele is about 2 times higher in Koreans than in Caucasians and Afro-Americans. These studies suggest that there is racial difference in the distribution of MCP-1 polymorphism. In Korean, MCP-1-2518A allele is minor allele and minor allele was associated with plasma MCP-1 level.40 MCP-1-increasing allele which is more sensitive to risk factors and are more prone to development of cardiovascular disease.

Genetic background may interact with supplementation and influence the effect of the supplement. Perez-Martinez et al.41 reported gene-nutrient interactions on phosphoenolpyruvate carboxykinase and insulin sensitivity. Among subjects with n-3 polyunsaturated fatty acid (PUFA) levels above the population median, carriers of the C/C genotype exhibited lower plasma concentrations of fasting insulin and model assessment of insulin resistance as compared with C/C carriers with n-3 PUFA below the median. And in another study,42 glucokinase regulatory protein genetic variant interacts with omega-3 PUFA in metabolic syndrome. In addition to longitudinal studies, gene variation effect in response to supplementation have shown among clinical trials, with response ranging from better than average to nonresponse or even adverse response.43 Oatmeal has been widely known for cholesterol-lowering effect.44 However, Ye et al.45 reported that response of serum LDL-C to oatmeal consumption depends on cytochrome P450 family 7 subfamily A member 1 gene rs3808607 (CYP7A1) genotype in Chinese. MacKay et al.43 also demonstrated CYP7A1 and apolipoprotein E (APOE) isoform are associated with the extent of reduction in circulating LDL-C in response to plant sterol consumption. More specifically, CYP7A1 T/T homozygotes showed no LDL-C lowering effect, whereas the presence of the G-allele associated with LDL-C response in a dose dependent fashion. Moreover, genoset CYP7A1 T/T/APOE ε3 was associated with non-responsiveness for plant sterol consumption.43 These results could serve as personalized markers to consumption.

Fewer studies have investigated the interaction between MCP-1 genotype and supplementation. In our previous study,21 the effects of spirulina supplementation were different depending on MCP-1 genotypes. In A/A genotype, spirulina supplementation resulted in significant changes for the plasma levels of MCP-1, IL-2; MCP-1 level was decreased, and the levels of IL-2 was increased. The concanavalin A- and phytohemagglutinin-induced mitogenesis was improved after spirulina supplementation. However, other genotype showed fewer effective results. The significantly increasing effect for IL-2 level was observed in G/A genotype and increasing effect for IL-2 level was only found in G/G genotype. In this study, for the subject with A allele, the plasma level of antioxidant nutrient such as vitamin A, and folate was significantly higher in spirulina group than placebo. The plasma TAS was significantly higher and the TBARS level was significantly lower in spirulina group than placebo. For the subject with G/G genotype, among the antioxidant nutrients, only folate level was significantly higher in spirulina group than placebo. The plasma TAS level was significantly higher in spirulina group with G/G genotype. These results suggest that personalized management might be needed in different genotype. Moreover, it is important to exclude the confounding factors of the results as much as possible by examining the subjects' basic lifestyle, eating habits, and dietary intake. In particular, dietary intake, alcohol consumption, and smoking can affect the blood antioxidant index regardless of supplementation. In this study, it was https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 8/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

confirmed that there was no difference in dietary intake, drinking and smoking according to genotype, and only the efficacy of supplements was examined.

One limitation of our trial was size (n = 78), which could be considered small for investigation of genetic associations. The evidence from this study is not enough to start making specific personalized nutritional recommendations based on genetic information, results from this study are valuable for further study to document the relationship between MCP-1 polymorphism and diet, and careful attention should be paid to subsequent studies to involve enough subjects for each phenotype and genotype with sufficient statistical power.

In conclusion, our data represent a beginning step in evaluating the use of common genetic variations to predict an individual’s response to supplementation. The efficacy of the spirulina supplement in reducing lipid peroxidation products was associated with MCP-1 allele, with A/A allele carriers being more responsive to spirulina supplement in reducing TBARS and increasing plasma folate level than G/G homozygous individuals. Our results demonstrate that the response of plasma antioxidant status to spirulina supplement has high interindividual variability, which is influenced by MCP-1 polymorphisms. These genetic variants could be used in the future to identify individuals who will benefit the most from spirulina intervention in terms of antioxidant capacity. The use of supplement, in the context of personalized nutritional recommendations, would greatly increase its efficacy in reducing risk factors of target disease.

REFERENCES

1. Egashira K. Molecular mechanisms mediating inflammation in vascular disease: special reference to monocyte chemoattractant protein-1. Hypertension 2003;41(3 Pt 2):834-41. PUBMED | CROSSREF 2. Rollins BJ. Chemokines. Blood 1997;90(3):909-28. PUBMED | CROSSREF 3. Nelken NA, Coughlin SR, Gordon D, Wilcox JN. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest 1991;88(4):1121-7. PUBMED | CROSSREF 4. Rollins BJ. Monocyte chemoattractant protein 1: a potential regulator of monocyte recruitment in inflammatory disease. Mol Med Today 1996;2(5):198-204. PUBMED | CROSSREF 5. Soejima H, Ogawa H, Yasue H, Kaikita K, Takazoe K, Nishiyama K, et al. Angiotensin-converting enzyme inhibition reduces monocyte chemoattractant protein-1 and tissue factor levels in patients with myocardial infarction. J Am Coll Cardiol 1999;34(4):983-8. PUBMED | CROSSREF 6. Aukrust P, Berge RK, Ueland T, Aaser E, Damås JK, Wikeby L, et al. Interaction between chemokines and oxidative stress: possible pathogenic role in acute coronary syndromes. J Am Coll Cardiol 2001;37(2):485-91. PUBMED | CROSSREF 7. Zhang PY, Xu X, Li XC. Cardiovascular diseases: oxidative damage and antioxidant protection. Eur Rev Med Pharmacol Sci 2014;18(20):3091-6. PUBMED 8. Aviram M. Review of human studies on oxidative damage and antioxidant protection related to cardiovascular diseases. Free Radic Res 2000;33 Suppl:S85-97. PUBMED 9. Kumar A, Shalmanova L, Hammad A, Christmas SE. Induction of IL-8(CXCL8) and MCP-1(CCL2) with oxidative stress and its inhibition with N-acetyl cysteine (NAC) in cell culture model using HK-2 cell. Transpl Immunol 2016;35:40-6. PUBMED | CROSSREF 10. Denev PN, Kratchanov CG, Ciz M, Lojek A, Kratchanova MG. Bioavailability and antioxidant activity of black chokeberry (Aronia melanocarpa) polyphenols, in vitro and in vivo evidences and possible mechanisms of action, a review. Compr Rev Food Sci Food Saf 2012;11(5):471-89. CROSSREF https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 9/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

11. Paulrayer A, Adithan A, Lee JH, Moon KH, Kim DG, Im SY, et al. Aronia melanocarpa (Black Chokeberry) reduces ethanol-induced gastric damage via regulation of HSP-70, NF-κB, and MCP-1 signaling. Int J Mol Sci 2017;18(6):1195-208. PUBMED | CROSSREF 12. Ko J, Kim K. Effects of exercise and diet composition on expression of MCP-1 and oxidative stress-related mRNA of adipose tissue in diet-induced obese mice. J Exerc Nutrition Biochem 2013;17(4):181-8. PUBMED | CROSSREF 13. Ahn N, Kim K. Combined influence of dietary restriction and treadmill running on MCP-1 and the expression of oxidative stress-related mRNA in the adipose tissue in obese mice. J Exerc Nutrition Biochem 2014;18(3):311-8. PUBMED | CROSSREF 14. Xu J, Liao YF, Zhou WP, Ming HL, Wang QH. The MCP-1 gene A-2518G polymorphism confers an increased risk of vascular complications in type 2 diabetes mellitus patients. Genet Test Mol Biomarkers 2015;19(8):411-7. PUBMED | CROSSREF 15. Jia LQ, Shen YC, Guo SJ, Hu QJ, Pang CS, Wang T, et al. The 2518 A/G polymorphism in the MCP-1 gene and cancer risk: a meta-analysis. Asian Pac J Cancer Prev 2013;14(6):3575-9. PUBMED | CROSSREF 16. Zhang Y, Zhang J, Zeng L, Huang H, Yang M, Fu X, et al. The -2518A/G polymorphism in the MCP-1 gene and tuberculosis risk: a meta-analysis. PLoS One 2012;7(7):e38918-24. PUBMED | CROSSREF 17. Altamura AC, Mundo E, Cattaneo E, Pozzoli S, Dell'osso B, Gennarelli M, et al. The MCP-1 gene (SCYA2) and mood disorders: preliminary results of a case-control association study. Neuroimmunomodulation 2010;17(2):126-31. PUBMED | CROSSREF 18. Mohseni H, Amani R, Hosseini SA, Ekrami A, Ahmadzadeh A, Latifi SM. Genetic variations in VDR could modulate the efficacy of vitamin D3 supplementation on inflammatory markers and total antioxidant capacity among breast cancer women: a randomized double blind controlled trial. Asian Pac J Cancer Prev 2019;20(7):2065-72. PUBMED | CROSSREF 19. Borel P, Desmarchelier C. Genetic variations associated with vitamin A status and vitamin A bioavailability. Nutrients 2017;9(3):246-62. PUBMED | CROSSREF 20. Dalan R, Goh LL, Lim CJ, Seneviratna A, Liew H, Seow CJ, et al. Impact of Vitamin E supplementation on vascular function in haptoglobin genotype stratified diabetes patients (EVAS trial): a randomised controlled trial. Nutr Diabetes 2020;10(1):13-26. PUBMED | CROSSREF 21. Park HJ, Lee HS. Monocyte chemoattractant protein-1 polymorphism interaction with spirulina immunomodulatory effects in healthy Korean elderly: a 16 week, double-blind randomized clinical trial. Nutr Res Pract 2017;11(4):290-9. PUBMED | CROSSREF 22. Belay A, Ota Y, Miyakawa K, Shimamatsu H. Current knowledge on potential health benefits of spirulina. J Appl Phycol 1993;5(2):235-41. CROSSREF 23. Kato T, Takemoto K, Katayama H, Kuwabara Y. Effects of spirulina on dietary hypercholesterolemia in rats. Nihon Eiyo Shokuryo Gakkai Shi 1984;37(4):323-32. CROSSREF 24. Ishii K, Katoh T, Okuwaki Y, Hayashi O. Influence of dietarySpirulina platensis on IgA level in human saliva. J Kagawa Nutr Univ 1999;30:27-33. 25. Qureshi MA, Kidd MT, Ali RA. Spirulina platensis extract enhances macrophages functions after in vitro exposure. J Nutr Immunol 1995;3:35-44. 26. Piñero Estrada JE, Bermejo Bescós P, Villar del Fresno AM. Antioxidant activity of different fractions of Spirulina platensis protean extract. Farmaco 2001;56(5-7):497-500. PUBMED | CROSSREF 27. Hirata T, Tanaka M, Ooike M, Tsunomura T, Sakaguchi M. Antioxidant activities of phycocyanobilin prepared from Spirulina platensis. J Appl Phycol 2000;12(3/5):435-9. CROSSREF 28. Kim WY, Park JY. The effect of spirulina on lipid , antioxidant capacity and immune function in Korean elderlies. Korean J Nutr 2003;36(3):287-97.

https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 10/11 MCP-1 Polymorphism and Spirulina Supplementation Benefits

29. Martínez-Sámano J, Torres-Montes de Oca A, Luqueño-Bocardo OI, Torres-Durán PV, Juárez-Oropeza MA. Spirulina maxima decreases endothelial damage and oxidative stress indicators in patients with systemic arterial hypertension: results from exploratory controlled clinical trial. Mar Drugs 2018;16(12):496-508. PUBMED | CROSSREF 30. Szulinska M, Gibas-Dorna M, Miller-Kasprzak E, Suliburska J, Miczke A, Walczak-Gałezewska M, et al. Spirulina maxima improves insulin sensitivity, lipid profile, and total antioxidant status in obese patients with well-treated hypertension: a randomized double-blind placebo-controlled study. Eur Rev Med Pharmacol Sci 2017;21(10):2473-81. PUBMED 31. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;18(6):499-502. PUBMED | CROSSREF 32. Yagi K. Assay for blood plasma or serum. In: Packer L, editor. Methods in Enzymology. New York, NY: Academic Press Inc.; 1984. 33. Benedetti S, Benvenuti F, Pagliarani S, Francogli S, Scoglio S, Canestrari F. Antioxidant properties of a novel phycocyanin extract from the blue-green alga Aphanizomenon flos-aquae. Life Sci 2004;75(19):2353-62. PUBMED | CROSSREF 34. Miranda MS, Cintra RG, Barros SB, Mancini Filho J. Antioxidant activity of the microalga Spirulina maxima. Braz J Med Biol Res 1998;31(8):1075-9. PUBMED | CROSSREF 35. Pola R, Flex A, Gaetani E, Proia AS, Papaleo P, Di Giorgio A, et al. Monocyte chemoattractant protein-1 (MCP-1) gene polymorphism and risk of Alzheimer's disease in Italians. Exp Gerontol 2004;39(8):1249-52. PUBMED | CROSSREF 36. Zietz B, Büchler C, Herfarth H, Müller-Ladner U, Spiegel D, Schölmerich J, et al. Caucasian patients with type 2 diabetes mellitus have elevated levels of monocyte chemoattractant protein-1 that are not influenced by the -2518 A-->G promoter polymorphism. Diabetes Obes Metab 2005;7(5):570-8. PUBMED | CROSSREF 37. Kim HL, Yang SH, Oh YK, Lee JE, Oh JE, Yoon HJ, et al. The effects of polymorphism in the MCP-1 gene regulatory region on MCP-1 expression and the manifestation of lupus nephritis. Korean J Nephrol 2002;21(1):137-44. 38. Pae CU, Kim JJ, Yu HS, Lee CU, Lee SJ, Jun TY, et al. MCP-1 promoter -2518 polymorphism may have an influence on clinical heterogeneity of in the Korean population. Neuropsychobiology 2004;49:111-4. PUBMED | CROSSREF 39. Hwang SY, Cho ML, Park B, Kim JY, Kim YH, Min DJ, et al. Allelic frequency of the MCP-1 promoter -2518 polymorphism in the Korean population and in Korean patients with rheumatoid arthritis, systemic lupus erythematosus and adult-onset Still's disease. Eur J Immunogenet 2002;29(5):413-6. PUBMED | CROSSREF 40. Park HJ. Association of MCP-1 polymorphism with cardiovascular disease risk factors in Korean elderly. J Nutr Health 2013;46(6):511-20. CROSSREF 41. Perez-Martinez P, Garcia-Rios A, Delgado-Lista J, Gjelstad IM, Gibney J, Kieć-Wilk B, et al. Gene-nutrient interactions on the phosphoenolpyruvate carboxykinase influence insulin sensitivity in metabolic syndrome subjects. Clin Nutr 2013;32(4):630-5. PUBMED | CROSSREF 42. Perez-Martinez P, Delgado-Lista J, Garcia-Rios A, Mc Monagle J, Gulseth HL, Ordovas JM, et al. Glucokinase regulatory protein genetic variant interacts with omega-3 PUFA to influence insulin resistance and inflammation in metabolic syndrome. PLoS One 2011;6(6):e20555-61. PUBMED | CROSSREF 43. MacKay DS, Eck PK, Gebauer SK, Baer DJ, Jones PJ. CYP7A1-rs3808607 and APOE isoform associate with LDL cholesterol lowering after plant sterol consumption in a randomized clinical trial. Am J Clin Nutr 2015;102(4):951-7. PUBMED | CROSSREF 44. Ho HV, Sievenpiper JL, Zurbau A, Blanco Mejia S, Jovanovski E, Au-Yeung F, et al. The effect of oat β-glucan on LDL-cholesterol, non-HDL-cholesterol and apoB for CVD risk reduction: a and meta-analysis of randomised-controlled trials. Br J Nutr 2016;116(8):1369-82. PUBMED | CROSSREF 45. Ye M, Sun J, Chen Y, Ren Q, Zhao Y, Pan Y, et al. Response of serum LDL cholesterol to oatmeal consumption depends on CYP7A1_rs3808607 genotype in Chinese. Asia Pac J Clin Nutr 2020;29(2):423-33. PUBMED https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e24 11/11