Methods

Middle-aged to elderly subjects were recruited from among consecutive visitors to the Anti-Aging Center at Ehime University Hospital from March 2006 to November 2012. They participated in the voluntary medical checkup program provided to residents of Ehime Prefecture, Japan, titled “Anti-Aging Doc,” which was specifically designed to evaluate age-related disorders including atherosclerosis, cardiovascular disease, physical function, and cognitive impairment. Of the 1,698 consecutive patients initially approached, 1,490 gave written consent to undergo all procedures and had no history of symptomatic cardiovascular events including peripheral arterial disease, stroke, coronary heart disease, and congestive heart failure. All participants were functionally independent in their daily lives. However, 59 subjects had plasma BNP levels of ≥100 pg/ml. Because these subjects may have had subclinical abnormalities, they were excluded from the analysis. Accordingly, 1,431 subjects were studied. The series of studies to which the present study belongs was approved by the Ethics Committee of Ehime University Graduate School of Medicine.

Thigh muscle cross-sectional area (CSA) was measured using computed tomography (LightSpeed VCT; GE Healthcare, Tokyo, Japan) at the mid-thigh, measured as the midpoint from the inguinal crease to the proximal pole of the patella. The muscle CSA (in square centimeters), excluding intramuscular fat, was computed using an attenuation range of 0 to 100 Hounsfield units. The visceral fat area was measured using computed tomography at the level of the umbilicus, with an attenuation range of −150 to −50 Hounsfield units. Images were obtained with a minimal slice width of 5 mm and analyzed using OsiriX software (OsiriX Foundation, Geneva, Switzerland).

Visceral obesity was defined as a visceral fat area of >100 cm ² in both men and women in accordance with the Japanese criteria defining metabolic syndrome. Sarcopenia was defined separately in men and women. Two criteria were used: values within 1 standard deviation (−1 SD) obtained from subjects aged <50 years and those whose values were in the lowest 20% of the study participants.

The pulse wave velocity was measured using a volume plethysmograph (PWV/ABI; Omron Healthcare Co., Ltd., Kyoto, Japan). A detailed explanation of this device and the validity and reproducibility of its measurements have been provided elsewhere. The brachial-ankle pulse wave velocity (baPWV) was calculated from the time interval between the wave fronts of the brachial and ankle waveforms (ΔTba) and the path length from the brachium to the ankle. The path length from the suprasternal notch to the brachium (Lb) or ankle (La) was obtained using the following formulas: Lb = 0.2195 × height + 2.0734 and La = 0.8129 × height + 12.328. The baPWV was then obtained using the equation (La − Lb)/ΔTba. The intra-measurement reproducibility and between-measurement reproducibility (coefficient of variation) of baPWV in our laboratory were 2.1% ± 1.8% and 2.2% ± 1.5%, respectively.

The radial waveform was analyzed in the left radial artery using an automated tonometric method (HEM-9000AI; Omron Healthcare Co., Ltd.) with subjects in the sitting position after at least 5 minutes of rest. The brachial blood pressure (BP) was measured simultaneously in the right brachium with an oscillometric device incorporated into the HEM-9000AI. The HEM-9000AI device is programed to automatically determine the pressure against the radial artery to obtain the optimal arterial waveform. The late systolic second peak BP (SBP2) was calculated by calibration with the brachial systolic BP (SBP). The pulse pressure (PP) was obtained by the formula PP = SBP − DBP, where DBP is the diastolic BP, and the PP2 was obtained from the formula SBP2 − DBP. The measurements were repeated twice and the mean values were obtained. The radial PP2 has been shown to accurately reflect the transfer function–derived aortic PP and was used as central BP-related values in the present study.

Plasma samples were obtained from each participant after an overnight fast. The samples were immediately frozen and stored at −80°C until measurements were taken. The plasma concentration of leptin was determined using a commercially available radioimmunoassay kit (Leptin HL-81K; Linco Research Inc., St. Charles, Missouri). Total plasma adiponectin levels, including high- to low-molecular-weight adiponectin, were measured using commercially available enzyme-linked immunosorbent assay kit systems (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). The plasma BNP concentration was measured using a standard chemiluminescent enzyme immunoassay (PATHFAST BNP assay kit; Mitsubishi Chemical Medience Corporation, Tokyo, Japan). The interassay reproducibility of leptin, adiponectin, and BNP (coefficient of variation) was 7.2%, 11.5%, and 3.9%, and the intra-assay reproducibility (for intra-assay variation) was 4.1%, 4.0%, and 4.3%, respectively. Plasma BNP, adiponectin, and leptin levels were analyzed by log transformation.

Lifestyle, medical history, and prescribed drugs were evaluated by questionnaire. Anthropometric measurements were performed by a trained nurse. Venous blood was collected for measurement of lipid, insulin, glucose, and high-sensitivity C-reactive protein levels. Homeostasis model assessment–insulin resistance was calculated as an index of insulin resistance. Renal function was evaluated based on the estimated glomerular filtration rate, which was calculated from the plasma creatinine values using the following formula specifically developed for the estimation of the glomerular filtration rate in Japanese subjects by the Japanese Society of Nephrology: 194 × creatinine ^–1.094 × age ^–0.287 × 0.739 (if female). Physical activity was assessed using a questionnaire that evaluated the subjects’ daily physical activities from the following categories: sufficient or almost sufficient, relatively insufficient, and insufficient.

Values are expressed as the mean ± SD unless otherwise specified. First, we compared the clinical backgrounds of the studied subjects divided into tertiles based on plasma BNP levels in men and women separately. Plasma BNP levels were further compared between tertiles of body mass index (BMI), visceral fat area, and thigh muscle CSA obtained in men and women separately. Multiple regression analyses were performed to ascertain whether muscle CSA and visceral fat area were associated with the plasma BNP level independently of other possible confounding parameters. Finally, the subjects were categorized into 4 groups based on the presence or absence of visceral obesity and sarcopenia using the definitions mentioned previously, and the plasma BNP levels were compared. Differences in numeric variables among groups were assessed using analysis of variance testing followed by Tukey’s correction for multiple comparisons; differences in frequency were assessed using the chi-square test. Corrections for age and other confounding parameters were made using these parameters in multiple regression analyses. All analyses were undertaken using commercially available statistics software (JMP version 10.0; SAS Institute, Cary, North Carolina). A p value of <0.05 was considered to be statistically significant.

Results

The clinical characteristics of the studied subjects (divided into men and women) by tertiles of BNP levels are listed in Table 1 . The simple correlation coefficients for the plasma BNP levels are summarized in Supplementary Table 1 .

The plasma BNP levels in the BMI, visceral fat area, and thigh muscle CSA tertiles are depicted in Figure 1 . The visceral fat area and thigh muscle CSA were significantly and negatively related to the plasma BNP level, whereas the BMI was not significantly associated with the plasma BNP level after correction for confounding parameters including the visceral fat area and thigh muscle CSA.

Plasma BNP levels in tertiles of visceral fat area (A and B) , thigh muscle CSA (C and D) , and BMI (E and F) . A , C , and E are not corrected. B , D , and F are corrected for potentially confounding factors including age, gender, mean BP, heart rate, lipid profile, homeostasis model assessment–insulin resistance, high-sensitivity C-reactive protein, estimated glomerular filtration rate, use of antihypertensive drugs, use of antidyslipidemic drugs, use of antidiabetic drugs, current smoking, and physical activity. The tertile of visceral fat area was further corrected for body height, body weight, and thigh muscle CSA. The tertile of thigh muscle CSA was further corrected for body height, body weight, and visceral fat area. The tertile of BMI was further corrected for visceral fat area and thigh muscle CSA. Values are represented as mean ± SEM. The number at the base of each column represents the number of subjects in each tertile. ∗p <0.05 versus tertile 1; ^† p <0.05 versus tertile 2.

Multiple regression analyses for BNP further demonstrated the independence of the association between thigh muscle CSA and the plasma BNP level ( Table 2 ). Analyses performed separately in men and women showed similar findings, although there were several gender-specific differences ( Supplementary Table 2 ). In men, the BNP level was associated with the adiponectin level but not with the leptin level. In women, the BNP level was more closely associated with the leptin than the adiponectin level.

Table 2

Multiple regression analyses of plasma levels of B-type natriuretic peptide

Variables	Model 1		Model 2
	β	p	β	p
N	1295		1295
Women = 1	0.10	0.052	0.02	0.55
Age (years)	0.31	<0.0001	0.28	<0.0001
Heart rate (bpm)	−0.14	<0.0001	−0.14	<0.0001
Mean blood pressure (mm Hg)	−0.02	0.66	−0.02	0.60
Body height (cm)	0.07	0.18
Body weight (kg)	0.21	0.0028
Body mass index (kg/m 2 )			0.07	0.15
Visceral fat area (cm 2 )	−0.07	0.11	−0.03	0.68
Thigh muscle cross sectional area (cm 2 )	−0.27	<0.0001	−0.17	0.0026
Interaction between sex*thigh muscle cross-sectional area	0.0	0.94	0.0	0.97
Adiponectin (μg/ml)	0.11	0.0003	0.12	0.0002
Leptin (pg/ml)	−0.07	0.055	−0.05	0.16
Brachial-ankle pulse wave velocity (cm/sec)	−0.06	0.09	−0.07	0.07
Radial pulse pressure 2 (mm Hg)	0.09	0.018	0.08	0.039
HOMA-IR	−0.04	0.21	−0.04	0.16
Total cholesterol (mg/dl)	−0.09	0.0022	−0.09	0.0018
High density lipoprotein cholesterol (mg/dl)	0.07	0.049	0.06	0.06
Triglyceride (mg/dl)	0.03	0.39	0.03	0.45
High sensitivity C-reactive peptide (mg/ml)	0.05	0.054	0.04	0.086
Estimated glomerular filtration ratio (ml/min/1.73 m 2 )	−0.04	0.12	−0.04	0.12
Current smoking (yes = 1)	0.01	0.68	0.01	0.58
Antihypertensive drugs (yes = 1)	0.03	0.31	0.03	0.33
Antidyslipidemic drugs (yes = 1)	−0.03	0.29	−0.03	0.29
Antidiabetic drugs (yes = 1)	−0.04	0.13	−0.04	0.14
Physical activity (insufficient = 1)	−0.02	0.45	−0.02	0.34

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