Daily Intake of a Commercially Available Vegetable Juice Has Beneficial Effects on Metabolic Syndrome-Related Biomarkers in Young , Healthy Japanese Adults

Adequate vegetable intake is important in reducing the risk of lifestyle-related diseases; however, intake amongst young Japanese adults is poor. This study aimed to assess the effect of daily intake of a commercially available vegetable juice on nutritional status and metabolic syndrome-related markers in healthy young adults. Fifty-one healthy adults (14 men, 37 women), age 20 22 years were recruited, and their food and nutritional intake was surveyed using an FFQ based on Food Groups. Subjects were divided into either a control group, who were not administered vegetable juice, or an intervention group, who consumed 200 mL vegetable juice daily for 2 months. Anthropometric data, serum carotenoid concentrations, blood and urinary parameters were measured at baseline and at 2 months. The average intake of vegetables in all subjects at baseline was approximately 140 g/day, less than half the dose recommended by the Japanese government. Total serum carotenoid concentration in the intervention group doubled from baseline after 2 months. In particular, serum concentrations of α-carotene, β-carotene, and lycopene were increased. In the intervention group, 24-hour urinary potassium excretion increased significantly, and 24-hour urinary sodium to potassium ratio decreased significantly. Waist circumference and systolic blood pressure also decreased significantly. Daily intake of a commercial vegetable juice for 2 months improved nutritional status in young adults with inadequate vegetable intake, shown by improved Corresponding author.


Introduction
Adequate intake of vegetables is very important in the prevention of lifestyle-related diseases [1].In Japan, the National Health Promotion Campaigns for the 21st Century (Healthy Japan 21), advocated by the Ministry of Health, Labour, and Welfare, recommends that adults eat more than 350 g of vegetables per day for maintenance of good health [2] [3].However, the average daily intake of vegetables for Japanese people is approximately 270 g.Intake is especially poor in young adults (between 20 and 29 years old), who eat approximately 230 g per day [4].Intake of green-yellow vegetables rich in carotenoids is especially inadequate in this age group.Daily intake of carotenoid rich vegetables has been estimated at approximately 60 g, only half of that recommended by Healthy Japan 21 [5].Based on these observations, increasing vegetable intake in young Japanese adults is of urgent priority in the fields of nutrition and public health.
Carotenoids are important antioxidants abundant in vegetables.They are thought to prevent lifestyle-related diseases through their antioxidant properties and the function of vitamin A (retinol), derived from provitamin A carotenoids such as β-carotene and β-cryptoxanthin [6] [7].Many epidemiological studies have revealed significant negative relationships between carotenoid intake or serum/plasma carotenoid levels and the risks of various lifestyle-related diseases [8]- [12].
Potassium is another important nutrient found in vegetables, and can also help to prevent lifestyle-related diseases.It contributes to the maintenance of normal blood pressure, and in the prevention of cardiovascular diseases [13] [14].In particular, the balance of sodium and potassium intake is crucial for the prevention of hypertension.Urinary sodium to potassium ratio is thought to be a more useful biomarker in the evaluation of hypertension incidence than either sodium or potassium alone [15].Intake of vegetables is known to be an effective means to improve urinary sodium to potassium ratio [16] [17].
Vegetable juice is thought to be a convenient alternative to the consumption of large amounts of vegetables, as it can still supply vital nutrients such as carotenoids and potassium.There are some reports that daily intake of vegetable juice increases dietary intake of such nutrients [18] [19], however the effects of vegetable juice on nutritional status and metabolic syndrome, a risk factor for lifestyle-related diseases such as cardiovascular disease, have not been extensively evaluated.In this study, we conducted an interventional study recruiting young Japanese adults, whose vegetable intake was thought to be inadequate.We investigated the effect of vegetable juice on serum carotenoid concentrations, urinary sodium to potassium ratio, and metabolic syndrome-related markers.

Subjects
The study protocols were approved by the Ethics Committee of Mukogawa Women's University and KAGOME CO., LTD., and were carried out in accordance with the International Ethical Guidelines and the Declaration of Helsinki.A total of 51 students from Hyogo University were recruited (14 men and 37 women), between 20 -22 years of age.All subjects provided written informed consent to this study.

Study Design
Subjects were surveyed regarding their food and nutritional intake using an FFQ based on Food Groups (FFQg) and "Excel Eiyo-kun FFQg software" (version 2.0; Kenpakusha, Tokyo, Japan).The questionnaire was completed during the 6 days prior to the intervention period, and the energy and nutrient intake of each subject were estimated based on 29 food groups and 10 kinds of cooking [20].Subsequently, subjects were divided in two groups: a control group (N = 20; 6 males and 14 females) and an intervention group (N = 31; 8 males and 23 females).The subjects in the intervention group consumed one pack of the vegetable juice daily (200 mL) for 2 months.No experimental food was applied to the control group throughout this study.Anthropometric data, blood, and urine samples were collected for all subjects at baseline and following the intervention period.

Anthropometric Measurements and Collection of Blood and Urine Samples
All subjects were asked to abstain from consuming any foods or drinks (other than water) after 9:00 pm on the day before anthropometric measurements and collection of blood and urine samples, until after the assessment.Height, body weight, and body fat ratio were measured with commonly available tools.Waist circumference was measured with a tape measure at the level of the navel.Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured with an automated blood pressure measurement system (HEM-907; OMRON, Kyoto, Japan).A blood sample was drawn from the antecubital vein for each patient, and the serum was used to determine serum carotenoid concentrations and blood parameters.A 24 h urine sample was collected using an aliquot cup [21] and was used to determine urine parameters.

Measurement of Serum Carotenoid Concentrations
Serum carotenoid levels (lutein, β-cryptoxanthin, α-carotene, β-carotene, and lycopene) were determined by HPLC according to previous methods [22]- [25].In brief, 200 µL serum was prepared and 10 µL ethanol containing trans-β-8'-apocarotenal (10 µM) were added to the serum as an internal standard.Subsequently, 1 mL ethanol and a solution of n-hexane and dichloromethane (4:1, v/v, 4 mL) were added, the mixture was centrifuged at 1087 × g for 10 min, and the supernatant (4 mL) was evaporated under nitrogen gas.The residue was dissolved in 0.2 mL solvent mixture (n-hexane/acetone/ethanol/toluene, 10:7:6:7, v/v/v/v), and was filtered with a 0.2 μm filter.HPLC analysis was performed using a C30 carotenoid column (250 × 4.6 mm, 5 μm; YMC, Wilmington, NC, USA) and a photodiode array detector (SPD-M10, Shimadzu, Kyoto, Japan) at a detecting wavelength of 460 nm.Mobile phase A (A) consisted of a 75:15:10 mixture of methanol:tert-butyl-methyl-ether:water.Mobile phase B (B) consisted of an 8:90:2 mixture of methanol:tert-butyl-methyl-ether:water.Pumps were programmed to perform the following gradient at a flow rate of 1 mL/min: start at 100% A, a 25 min linear gradient to 100% B, 3 min at 100% B, a 2 min gradient back to 100% A, and 10 min at 100% A. Under these conditions, the concentrations of carotenoids were measured and corrected using an internal standard.

Statistical Analysis
All values are represented as mean ± standard deviation.Paired t-tests were used for comparisons within each group before and after the intervention.Data were analyzed using SPSS for Windows version 15.0 (SPSS Japan Inc, Tokyo, Japan).Values of p < 0.05 were accepted as significant.

Subjects
Of the 51 recruited participants, 38 completed the study (15/20 in the control group, 23/31 in the intervention group).Of the participants who failed to complete the study, 2 subjects (1 control, 1 intervention) were lost to follow-up after the intervention period, 10 (4 control, 6, intervention) did not complete urine sampling, and 1 subject in the intervention group was omitted from data analysis due to poor compliance in drinking vegetable juice (Figure 1).

Food and Nutritional Intake
The average daily intake of vegetables and green-yellow vegetables in all subjects were approximately 140 g and 50 g, respectively (Table 2).The average intakes of major nutrients (vitamins, minerals, and dietary fiber) were calculated using the FFQg and are also described in Table 2.

Anthropometric Data
No significant changes were observed when participants in the control group were compared before and after the intervention period.In contrast, continual intake of the vegetable juice for 2 months significantly decreased waist circumference and SBP (p = 0.006 and p = 0.034, respectively) (Table 4).

Blood Parameters
The blood parameters in the both groups at baseline and after the intervention period are shown in Table 5.
Among the parameters related to lipid metabolism, LDL-C and Total-C levels were significantly increased in the  intervention group after 2 months, however these changes were also observed in the control group.Among the parameters related to glucose metabolism, fasting blood glucose was significantly decreased in the intervention group (p = 0.019) and HbA1c level was significantly increased in the control group (p = 0.015).Folic acid level was significantly increased in both the control and intervention groups (p = 0.010 and p < 0.001, respectively).Homocysteine level was significantly increased in the control group (p = 0.010).Hemoglobin and hematocrit levels were significantly increased in the intervention group (p = 0.006 and p = 0.037, respectively).

Urine Parameters
The daily sodium excretion amount was not significantly different before and after the intervention period in the both groups.In contrast, daily urinary potassium excretion was significantly increased, and the urinary sodium to potassium ratio was significantly decreased in the intervention group after the intervention period (p < 0.001 and p < 0.001, respectively) (Table 6).

Discussion
Our study strongly confirmed that young Japanese adults consume inadequate amounts of vegetables relative to current public recommendations [2] [4] [5].The average vegetable intake of all subjects was approximately 140 g/day and this was surprisingly only 40% of the amount recommended (350 g/day) by the Japanese government [2].Moreover, intake of green-yellow vegetables in the subjects (50 g/day) was also about 40% of the recommendation amount (120 g/day) [2].This result is consistent with a previous report demonstrating that the average intake of green-yellow vegetables in young unmarried subjects was only 50% of the recommended level [5].
The "Dietary Reference Intakes (DRIs) for Japanese" proposes reference values for desirable dietary energy and nutrient intake by Japanese people to maintain and promote their health [28].Compared with the "DRIs for Japanese" [28], the intake of vitamin A, vitamin C, calcium, magnesium, and dietary fiber in our study subjects were less than 70% of the recommended amounts.These deficiencies likely reflect inadequate vegetable intake in the subjects.Potassium intake was more than 80% of the amount recommended in Japan (2500 mg/day for men, 2000 mg/day for women).However, this was not adequate compared to the recommendation by the WHO (at least 90 mmol/day (3510 mg/day) for adults) to reduce blood pressure and to decrease risk of cardiovascular diseases [29].
The total average serum carotenoid in the subjects before the intervention period was less than 1.5 μM and was remarkably lower than the levels recently reported in Japan [30]- [34].The daily intake of vegetable juice significantly increased total serum carotenoid concentration to approximately 3.5 μM, more than twice that of the baseline level.In particular, serum concentrations of α-carotene, β-carotene, and lycopene were increased.These increases were most likely due to the interventional vegetable juice, which was high in carotenoids.Donaldson established a carotenoid health index by analyzing 62 studies of plasma carotenoids and health outcomes, and proposed risk categories as follows: "very high risk": <1.0 μM, "high risk": 1.0 -1.5 μM, "moderate risk": 1.5 -2.5 μM, "low risk": 2.5 -4.0 μM, and "very low risk": >4.0 μM [9].In this study, of the 29 subjects whom completed vegetable juice supplementation and blood sampling, 23 (79%) were categorized in "very high risk" or "high risk" group at baseline, however only 2 (7%) remained in this group following the intervention period.Similarly, only 2 (7%) were categorized as "low risk" or "very low risk" before the intervention, and this increased to 25 (86%) following intervention.These results suggest that daily consumption of a commercially available vegetable juice may be sufficient to maintain an adequate serum carotenoid level.Previously, the cross-sectional INTERMAP study surveyed nutrient intake among individuals from China, Japan, the UK, and US, and assessed possible relationships between dietary patterns and risk of circulatory diseases.36 The study showed that individuals from Japan and China had a higher urinary sodium to potassium ratio than that of Western people, and it was postulated that this was likely to have adverse effects on blood pressure and higher mortality due to stroke.The average baseline urinary sodium to potassium ratio in this study (Men; 5.2, Women; 4.9) was marginally higher than that of Japanese adults in the INTERMAP study (Men; 4.5, Women; 4.1) [35].In the intervention group, urinary potassium excretion was significantly increased and urinary sodium to potassium ratio was significantly decreased after the intervention period.Moreover, SBP levels were significantly decreased in the intervention group, corresponding to the reduction in urinary sodium to potassium ratio.The vegetable juice used in this study contained 730 mg of potassium per 200 mL, and daily intake was therefore likely to increase urinary potassium concentration, improve sodium to potassium ratio, and reduce SBP in the intervention group.The presence of other anti-hypertensive compounds may also have contributed to these findings.Tomato, the most common raw material in the vegetable juice, contains significant quantities of γ-aminobutyric acid (GABA) [36], which has been shown to exhibit anti-hypertensive effects in human intervention studies [37]- [39].This may also provide a mechanism contributing to decreased SBP resulting from juice supplementation.
Previous studies have shown that lycopene and its metabolites can modulate lipid metabolism [40]- [42].Moreover, a recent human interventional study showed that tomato juice supplementation reduced waist circumference and serum levels of cholesterol and inflammatory adipokines associated with increased serum lycopene levels [43].We also observed a significant reduction in waist circumference and a concomitant increase in serum lycopene in the intervention group.The lycopene dose in this study (approximately 12 mg) was less than half of that in the previous interventional study (more than 30 mg).However, the significant increase in serum lycopene may have contributed to the reduction in waist circumference.Larger scale interventional studies are required to confirm the effect of lycopene on waist circumference and to clarify the underlying mechanism.
Daily supplementation with vegetable juice significantly decreased fasting blood glucose, and the increase in HbA1c observed in the control group was suppressed in the intervention group.Vegetables contain large amounts of dietary fiber, which is known to attenuate postprandial blood glucose elevation [44] [45].Recently, we revealed that consumption of a commercially available vegetable juice prior to, or concomitantly with a meal also exerted similar effects on postprandial blood glucose [46].Postprandial glycemic control during the day is important to control fasting glycemia and to prevent insulin resistance [47].The daily consumption of vegetable juice may be beneficial in the prevention of diabetes by facilitating glycemic control.
Homocysteine, a derivative of methionine, has been confirmed as a risk factor for ischemic heart disease and other vascular disorders [48].In contrast, folic acid is known to reduce homocysteine levels by promoting the conversion from homocysteine to methionine [49] [50].The experimental vegetable juice contained folic acid, although the content was variable (nutritional label: 13 to 110 μg/200mL), and an increase of serum folic acid was observed, particularly in the intervention group.This may explain why an increase in serum homocysteine levels was observed only in the control group.
Interestingly, vegetable juice supplementation increased hemoglobin and hematocrit levels, which are indicators of anemia.A human interventional study showed that vitamin A improved nonheme iron absorption from rice, wheat and corn, and the effect was also exerted by β-carotene, a major provitamin A carotenoid [51].Provitamin A carotenoid-rich vegetables were also shown to increase hemoglobin levels and decrease anemia rate with no effect on iron deficiency [52].Daily intake of vegetable juice might therefore be expected to be beneficial on the reduction of anemia although the relevant mechanisms have not been established.

Conclusion
We conducted an interventional study assessing the effects of a commercially available vegetable juice on the nutritional status of young Japanese adults, whose vegetable intake was thought to be inadequate.Daily intake of the vegetable juice for 2 months beneficially modulated markers of metabolic syndrome such as waist circumference, urinary sodium to potassium ratio, and serum homocysteine.Concomitant increases in serum carotenoids, urine potassium, and serum folic acid were observed.These results suggest that vegetable juice consumption elicits multiple benefits in the prevention of metabolic syndrome and lifestyle related diseases.

Figure 1 .
Figure 1.A flow diagram of participant recruitment is shown, including enrollment, grouping by control or intervention, and reasons for incomplete data.

Table 1 .
Nutritional information of the vegetable juice used in this study.
a Values obtained from nutritional labeling of the commercial vegetable juice.b Values obtained using HPLC.

Table 2 .
Vegetable and nutrient intake is shown for study participants at baseline.
Data is show as mean ± standard deviation (SD).RAE: retinol activity equivalents.

Table 3 .
Serum carotenoid concentrations are shown at baseline and after the interventional period.

Table 4 .
Anthropometric data are shown at baseline and after the interventional period.

Table 5 .
Blood parameters are shown at baseline and after the interventional period.

Table 6 .
Urine parameters are shown at baseline and after the interventional period.Data is shown as mean ± standard deviation (SD).Baseline vs.After 2 months, paired t-tests, *** p < 0.001.