Cardiovascular disease is the leading cause of mortality worldwide. The low-sodium diet approach, known as the Dietary Approaches to Stop Hypertension (DASH), and Washoku, the Japanese traditional dietary pattern, have demonstrated positive effects in preventing future cardiovascular disease.^1-4 If indices are used to prevent lifestyle diseases, we can easily judge whether daily dietary habits are appropriate or require revision. However, currently, there are no effective tools for this purpose.

The levels of several inflammatory factors are significantly lower in individuals with healthy dietary habits (e.g., high vegetable and fruit consumption) than in those with unhealthy dietary habits (e.g., sugar consumption, Western diet).^5,6 Pentraxin 3 (PTX3) is a known cardioprotective and anti-inflammatory factor. We previously reported that PTX3 levels are lower in overweight and obese individuals than individuals within the normal weight range.⁷ Previous studies reported a significant increase in plasma PTX3 concentrations in overweight and obese individuals who experienced dramatic weight loss.^8-10 In our recent study, we demonstrated that the decrease in plasma PTX3 levels positively correlated with body mass reduction in overweight or obese individuals, and this was less affected by habitual exercise compared with weight loss.¹⁰ We also showed that the group with increased circulating levels of PTX3 after weight loss from diet modification had lower arterial stiffness while decreased or unchanged levels were observed in Japanese overweight and obese adult men.¹¹ Therefore, PTX3 can be used as an index to determine whether dietary habits are appropriate for preventing cardiovascular diseases in obese individuals. Previous studies have revealed that plasma PTX3 levels are dependent on several factors, such as age, sex, obesity levels, exercise habits, and degree of weight loss.^7,8,10,12,13 However, there is no information on the effect of specific foods or dietary habits on plasma PTX3.

In the present study, we hypothesized that circulating PTX3 levels are lower in individuals with high sweet and sugar consumption. We investigated whether the specific intake frequency of food or nutrition had a significant effect on circulating PTX3 levels in Japanese adults.

Subjects and experimental design

We performed anthropometric measurements and blood pressure, arterial stiffness, and blood chemistry analyses on 485 adult men (62±11 years) and women (63±6 years). We also distributed a food frequency questionnaire (FFQ) to the participants. After excluding patients with insufficient data, patients taking medication, and those undergoing medical therapy, 327 individuals were selected for data analysis. This study was reviewed and approved by the Institutional Review Board of the University of Tsukuba (approval no. 27-68) and Ryutsu Keizai University (approval no. 7; January 13, 2015). All potential risks and procedures of the study were explained to the participants, and all participants provided written informed consent for participation. All measurements were performed between 8:00 AM and 12:00 AM after an overnight fast and included abstinence from alcohol, caffeine, and water. Participants also refrained from intense physical activity (exercise) for 24 hours before measurements were taken. The participants were evaluated in a supine resting condition in a quiet temperature-controlled room (24 to 26 °C). All measurements were performed after at least 15 minutes of rest.

Anthropometric measurement

Body weight was measured to the nearest 0.1 kg using a digital scale. Height was measured to the nearest 0.1 cm using a wall-mounted stadiometer. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m). Waist circumference (WC) was measured to the nearest 0.1 cm at the level of the umbilicus with participants in the standing position.

Blood pressure

Systolic blood pressure (SBP) and diastolic blood pressure were recorded from the left arm of participants in the supine position using a semi-automated device (PWV/ABI; Colin Medical Technology), as described in a previous study.¹⁰ Heart rate (HR) was measured using a semi-automated device (PWV/ABI).

Blood biochemistry

Blood samples (approximately 30 mL of whole blood) were collected from the antecubital vein after an overnight fast. Each blood sample was placed in a chilled tube containing ethylenediaminetetraacetic acid (2 mg/mL) and centrifuged at 2,000 ×g for 15 minutes at 4 °C. Plasma PTX3 concentrations were determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc.) as described previously.^7,8,10,13 The total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and glycosylated hemoglobin (HbA1c) concentrations and plasma concentrations of fasting glucose were determined using standard enzymatic techniques.

Food frequency questionnaire assessment

The FFQ, a semi-quantitative dietary survey that is widely used to assess food or nutrient intake in dietary studies, was assessed using analysis software (Excel Eiyoukun version 6.0; Kenpakusha).¹⁴ We used a version of the FFQ based on food group (FFQg), modified for use and validated in Japan, to obtain daily records for evaluating several food groups and each nutrient separately.^15-17 Participants responded to questions regarding their average intake frequency of foods prepared by several kinds of cooking methods and 29 food items and beverages over the last month before the study. Most food items were assessed using five frequency categories: never or seldom, 1–2 times/month, 1–2 times/week, 3–4 times/week, and almost daily. Beverage consumption was assessed using five frequency categories: almost never, 1–2 cups/month, 1–2 cups/week, 3–4 cups/week, and almost daily for green tea, tea, oolong tea, and coffee. The frequency of alcohol consumption was assessed using the following categories: never, former, and current drinkers (<1 time/week, 1–2 times/week, 3–4 times/week, and almost daily). The average daily nutrient intake was calculated by multiplying the frequency of consumption of each food item by the nutrient content per serving (SV). Total energy and nutrient intake were estimated using the seventh edition of the Japan Food Table.¹⁸ We used the number of food groups estimated using the answers on the analysis questionnaire, as listed in Table 1. Specifically, we used the Japanese Food Guide Spinning Top (JFGST), six food groups (6C), 13 food groups (13C), and 18 food groups (18C). The JFGST consists of five food groups: staple foods (rice, bread, noodles, and pasta), vegetable-based side dishes (including salads, cooked vegetables, and soups), main dishes (including fish, eggs, and meat), milk or dairy products, fruits, and confectionary and sugar-sweetened beverages (CSSB), with SV as the measurement unit.¹⁹ The other food groups were categorized based on 18C and consisted of grains, potatoes, green and yellow vegetables, other vegetables, Tsukudani (Japanese preserved food, which is boiled with sugar and salt), seaweed, beans, fish, meat, eggs, milk and dairy products, fruit, CSSB, sugar, nuts, fats and oils, and seasonings and spices. The 6C category included the following food groups: group 1 (fish, meat, eggs, and beans); group 2 (milk and dairy products, seaweed, and small fish); group 3 (green and yellow vegetables); group 4 (other vegetables, fungi, and fruits); group 5 (grains, potatoes, sugar, and CSSB); and group 6 (fats and oils). In these food categories, beans include bean products, such as boiled beans and tofu; fresh fish and vegetables include spinach, carrots, pumpkin, tomatoes, cabbage, and head lettuce; and fruits include citrus fruits. The measurement unit for the foods in 6C, 13C, and 18C was kcal. We used the following nutrient data estimated using the answers on the questionnaire: energy (kcal), protein (g), fat (g), carbohydrate (g), Na (mg), K (mg), Ca (mg), Mg (mg), P (mg), Fe (mg), Zn (mg), Cu (mg), Mn (mg), cryptoxanthin (µg), equivalence of β-carotene (µg), equivalence of retinol (µg), vitamin D (µg), equivalence of tocopherol (mg), vitamin K (µg), vitamin B1(mg), vitamin B2 (mg), niacin (mgNE), vitamin B6 (mg), vitamin B12 (µg), folic acid (µg), pantothenic acid (mg), vitamin C (mg), saturated fatty acids (g), monounsaturated fatty acids (g), polyunsaturated fatty acids (g), cholesterol (mg), total amount of dietary fiber (g), salt (g), n-3 fatty acid (g), and n-6 fatty acid (g).

Statistical analyses

We categorized all the participants into two groups based on their CSSB intake: the “low” (<200 kcal/week) and “high” (≥200 kcal/week) group; the groups were categorized using the threshold recommended by the Ministry of Agriculture, Forestry, and Fisheries in Japan.²⁰ The association of PTX3 levels and other covariants (physical and nutritional characteristics) were determined by the Least Absolute Shrinkage and Selection Operator (LASSO) analysis.²¹ This determines explanatory factors even if there are multicollinear or multivariant factors (i.e., binary and continuous variants). Model selection was conducted using the “glmnet” package within the R. We also applied a LASSO analysis to determine which parameter is associated with circulating PTX3 concentrations in age- or gender-limited groups. We analyzed four groups: men (n=193), women (n=134), men and women aged <65 years (n=162), and older men and women (>65 years; n=165). All data are reported as mean±standard deviation. Statistical significance was set a priori at P<0.05 for all comparisons.

As shown in Fig. 1, the recommended upper limit of CSSB intake for humans is 200 kcal/day. In our study, 192 and 135 participants were in the high and low CSSB intake groups, respectively. Table 2 shows the association of plasma PTX3 concentrations with physical and food characteristics of the participants. PTX3 was associated with sex (4.9%), Tsukudani (0.71%), CSSB (0.42%), WC (0.39%), niacin (0.19%), HDL-C (0.17%), HR (0.14%), age (0.13%), vitamin D (0.12%), and height (0.09%); the percentages indicate how much each factor affects PTX3 levels. The calculated PTX3 with the characteristic factors listed in Table 2 is shown in Fig. 2 (R=0.420, R²=0.176).

Because biological sex and age are known to affect PTX3 levels, we analyzed the characteristics that may also affect them in both biological sex and age groups (Table 2). Height (0.34%) and Tsukudani (0.39%) were significantly negatively associated with PTX3 levels in men. BMI (1.86%), WC (0.13%), and HDL-C (0.13%) were significantly associated with PTX3 levels in women. WC (0.52%) and Tsukudani score (0.48%) were significantly associated with PTX3 levels in men and women aged <65 years. In older individuals, Tsukudani (0.86%), HDL-C (0.78%), HR (0.45%), saturated fatty acid (0.31%), SBP (0.19%), and CSSB (0.12%) levels were associated with PTX3 levels.

In this study, we investigated the effect of food intake on circulating PTX3 levels in 327 Japanese men and women, based on their responses to a food frequency consumption questionnaire. Our results showed that men and women with a daily CSSB intake >200 kcal exhibited low plasma PTX3 levels. We first found by LASSO analysis that circulating PTX3 levels are affected by sex differences, dietary habits such as Tsukudani intake amount, CSSB, and physical characteristics (WC and HDL-C). In the analysis limited to individuals aged >65 years, PTX3 was significantly positively associated with HDL-C and HR, whereas it was negatively associated with SBP, Tsukudani, and CSSB intake. Moreover, PTX3 levels were associated with BMI, WC, and HDL-C in women. This study is the first to demonstrate that circulating PTX3 levels are affected by daily dietary habits and nutritional intake, as well as biological sex and age.

A previous cross-sectional study from a survey of 1,749 healthy Japanese individuals demonstrated that blood concentrations of PTX3 were dependent on age and sex.¹² Our results showed that predicted PTX3 levels can be explained with some factors (i.e., sex, WC, Tsukudani, and CSSB intake) by LASSO analysis. The negative relationship between PTX3 expression and obesity grade was similar to that observed in our previous studies.^7,8 However, in the present study, different results were obtained in other age groups or biological sex groups based on the relationship between PTX3 levels and WC. A previous cohort cross-sectional study of 10,899 Nordic men and women 40 years and older showed that compared with younger individuals, older individuals consumed significantly lower amounts of meat and sweets.²² Age- and sex-dependent dietary patterns have also been observed in Japanese cohort studies, and high-bread, low-rice, or high-vegetable dietary patterns have been identified in older women.²³ Based on these data, the positive relationship between plasma PTX3 levels and age in the present study was attributed to age-dependent dietary patterns. Notably, our results showed that the intake of Tsukudani, CSSB, and saturated fatty acids seemed to determine plasma PTX3 levels. Furthermore, the intake of Tsukudani and CSSB was significant determinants of PTX3 in individuals aged >65 years, but not in women. Saturated fatty acids seemed to be one of the determinants of PTX3 levels in the older group but not in the younger group. In women, BMI was a strong determinant of PTX3 levels; however, no nutritional factors were associated with PTX3 levels. The analysis showed that plasma PTX3 levels are most affected by sex and suggested that PTX3 levels are higher in women, which is similar to the results of Yamasaki et al.¹² Taken together, dietary habit trends differ depending on gender or age; therefore, they may partly affect and differ from physiological PTX3 grade in terms of generation or biological gender.

We previously reported that plasma PTX3 concentrations are significantly higher in young endurance-trained men than in untrained men of the same age, which showed a significant positive correlation between circulating PTX3 levels and HDL-C.¹⁰ This result was recently confirmed in a large-scale Korean study, which reported a significant positive relationship between PTX3 levels and HDL-C and a significantly negative relationship between PTX3 levels and TG.²⁴ In addition, PTX3 expression in visceral fat was significantly related to the ratio of LDL-C and HDL-C.²⁵ However, plasma PTX3 levels may be affected by ethnic or genetic differences, as PTX3 levels are significantly higher in Caucasian compared with African populations.²⁶ HDL-C induces PTX3 production by vascular endothelial cells, which bind to apoptotic cells and promote phagocytosis and a decrease in the atherosclerosis area, resulting in a cardioprotective effect.²⁷ In the present study, we observed a significantly positive relationship between plasma PTX3 levels and HDL-C, which was the 6th strongest factor of predicting PTX3 levels in the analysis of all subjects. In the group of men and women aged >65 years, the determinants of PTX3 levels were HDL-C, SBP, and HR. Our results showed that BMI in women and WC in the <65 years group was significant determinants of PTX3 levels. Previous data showed that PTX3 levels are lower in individuals with metabolic syndrome (MetS) than in individuals without MetS.¹² Taken together, these findings indicate that dietary habits that induce obesity and MetS result in lower PTX3 levels.

We previously showed that weight loss positively correlates to increased plasma PTX3.¹⁰ A weight loss intervention study targeting obese individuals indicated that PTX3 concentrations increase with weight loss in a manner that correlates with improvements in insulin sensitivity.²⁸ Patients with diabetes mellitus have lower circulating PTX3 levels and significantly lower renal glycosuria levels than healthy overweight individuals.²⁹ In an animal study, oral glucose tolerance test revealed that rats had low plasma PTX3 levels with abnormal glucose metabolism after being loaded with high fat and sugar.³⁰ In our previous study, we showed a significant positive relationship between the protein expression of PTX3 and glycosyltransferase 4, a cellular glucose transporter.³¹ These animal studies suggested that PTX3 levels are sensitive to changes in glucose metabolism. In the present study, we showed that a higher intake of Tsukudani, CSSB and saturated fatty acids resulted in lower plasma PTX3 levels in the general Japanese population, as observed in our cohort of 327 individuals. Interestingly, the traditional Japanese high-sugar food “Tsukudani” was one of the factors strongly associated with low plasma PTX3 levels. To the best of our knowledge, this is the first study to investigate the relationship between dietary habits and plasma PTX3 levels in the general population. Our findings indicate that high-sugar and high-fat eating habits result in low PTX3 levels.

Our study had several limitations. First, we did not evaluate the effects of daily physical activity on PTX3 levels using an activity meter. Therefore, we could not determine which dietary habits or physical activity levels had a much larger effect on modulating PTX3 levels in the general population. Second, our data did not reveal the precise physiological and metabolic mechanisms by which lifestyle factors are the strongest triggers that modulate PTX3 levels. However, the observation that plasma PTX3 levels are affected by biological sex, age, and dietary habits is a novel and significant finding.

The authors declare no conflict of interest.

This work was supported by Grant-in-Aid for Scientific Research 15K16519 from the Japan Society for the Promotion of Science.

Study concept and design: AZM; acquisition of data: HK, KT, and TO; analysis and interpretation of data: AZM; drafting of the manuscript: AZM and HK; critical revision of the manuscript: TO; statistical analysis: AZM and HZ; obtained funding: AZM; administrative, technical, or material support: TO and SM; and study supervision: TO and SM.