Subjects
A total of 225 subjects (94 males and 131 females: aged 51 to 85 years) received a population-based health examination in Uku town, a fishing community in southwestern Japan in 2019. This town is an isolated island in Sasebo city, located in Nagasaki prefecture, and the total population is about 2100. A detailed content of the recent survey in the same district was previously described [13, 14].
Data collection
Height and weight were measured, and body mass index (BMI) was calculated as weight (kg) divided by the square of height (m2) as an index of the presence or absence of obesity. Waist circumference was measured at the level of the umbilicus in a standing position. Blood pressure (BP) was measured in the right arm twice with a mercury sphygmomanometer after the subject had rested in the sitting (first) and supine (second) position for more than 5 min. Vigorous physical activity and smoking were avoided for at least 30 min before BP measurements. The second BP with the fifth phase diastolic pressure was used for analysis.
Blood was drawn from the antecubital vein for determinations of lipids profiles (total cholesterol, LDL-C, HDL-C, triglycerides, RLP-C, and lipoprotein(a) [Lp(a)]), apolipoproteins (apo A-I, apo B, apo C-III, and apo E), blood urea nitrogen (BUN), creatinine, uric acid (UA), fasting plasma glucose (FPG), insulin, and glycated hemoglobin A1c [HbA1c (NGSP)] in a morning after 12-h fasting. All chemistries were measured at commercially available laboratories (SRL Inc. Laboratory, Fukuoka, Japan, and The Kyodo Igaku Laboratory, Fukuoka, Japan). SRL Inc. Laboratory measured serum Lp(a) by latex immunoassay (LIA) method (SEKISUI Medical, Tokyo, Japan) [15], apolipoproteins by turbidimetric immunoassay method (SEKISUI Medical, Tokyo, Japan), and directly measured RLP-C by an immuno-separation technique (using an immunoaffinity gel containing monoclonal antibodies to human apo B-100 and apo A-I) (MiNARis medical, Tokyo, Japan) [16]. Intra- and inter-assay coefficients of variation of RLP-C in the commercially available laboratory (SRL inc. Laboratory, Fukuoka, Japan) were 7.6% and 7.8%, respectively. Other chemicals were examined in The Kyodo Igaku Laboratory, Fukuoka, Japan.
Fasting blood samples were centrifuged within 1 h after the collection. Estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease (MDRD) study equation modified with a Japanese coefficient [17]. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated from FPG and insulin levels [FPG (mg/dL) × insulin (μU/mL)/405] as a marker of IR [18]. Estimated RLP-C levels were defined as the following formula [total cholesterol – (LDL-C) − (HDL-C)]. The cut-off value of RLP-C was defined as greater than 7.5 mg/dL or not according to guidance of the SRL Inc. Laboratory [19].
Eating pattern was evaluated by a brief-type self-administered diet history questionnaire (BDHQ) [20]. The BDHQ is a four-page, fixed-portion questionnaire that asks about the consumption frequency of selected foods, but not about portion size, to estimate the dietary intake of fifty-eight food and beverage items during the preceding month. To facilitate reading and completion for the elderly, we used a large-print version, which increased the size to ten pages but that contained no other changes to structure or content. Details of the BDHQ’s structure, method of calculating dietary intake, and validity for food group and nutrient intakes among the adult population (31–76 years) have been described elsewhere [21, 22]. Briefly, the BDHQ consists of the following five sections: (i) intake frequency of food and non-alcoholic beverage items; (ii) daily intake of rice and miso soup; (iii) frequency of drinking and amount per drink for alcoholic beverages; (iv) usual cooking methods; and (v) general dietary behavior. Food and beverage items contained in the BDHQ were selected from foods commonly consumed in Japan, mainly from a food list used in the National Health and Nutrition Survey of Japan, while standard portion sizes were derived from several recipe books for Japanese dishes [22]. Information on dietary supplements was obtained only for total frequency of use, without specific names or types and quantity of the supplements. Estimates of the intake for fifty-eight food and beverage items were calculated using an ad hoc computer algorithm for the BDHQ [22].
Statistical analysis
Because of skewed distributions, the natural logarithmic transformation was performed for RLP-C, Lp(a), insulin, HOMA-IR, and triglycerides. Mean values, upper and lower 95% confidence limits, were exponentiated and presented geometric mean ± standard deviation (SD), where the SD was approximated as the difference of the exponentiated confidence limits divided by 3.92, the number of SD in a 95% confidence interval for normally distributed data. Chi-square tests were used for evaluation of categorical parameters. Uni- and multiple linear regression analyses adjusted for age and sex were used. Using some significant factors from multivariate linear regression analysis adjusted for age and sex, we performed the multiple stepwise regression analysis to see the strength and independency for estimated RLP-C. ROC analysis was performed to evaluate diagnostic ability of estimated RLP-C and HOMA-IR on dichotomized RLP-C (≥ 7.5 mg/dL or < 7.5 mg/dL). The C-statistics was reported as a measure of diagnostic ability, and Youden’s index was used to identify cut-off point on the ROC curve, which was defined as the maximum of (sensitivity + specificity-1).
P values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS version 26.0 (IBM Inc., Chicago, IL, USA).
