5.1 Data Source
This study was a secondary analysis of data from The National Health and Nutrition Examination Survey (NHANES) database, which was collected by the Centers for Disease Control and Prevention (CDC), National Center for Health Statistics (NCHS) in the USA. (http://www.cdc.gov/nchs/nhanes/). The NHANES program began in the United States in the early 1960s, and has been conducted as a series of surveys focusing on different population groups and health topics. Samples for the NHANES surveys are selected to represent the United States population of all ages. NHANES used a multi-stage, stratified, clustered, probability sampling design to identify a nationally representative sample of non-institutionalized civilians in the US Weights are created in NHANES to account for the complex survey design (including oversampling), survey non-response, and post-stratification adjustment to match total population counts from the Census Bureau. When a sample is weighted in NHANES, it is representative of the US civilian non-institutionalized resident population. A sample weight is assigned to each sample person. Further information about background, design, and protocols of the NHANES are available on the NHANES website (http://wwwn.cdc.gov/nchs/nhanes).
5.2 Ethics Statement
NHANES was reviewed and approved through the NCHS Research Ethics Review Board, and informed consent was provided by each participant. Please check the NHANES website for NCHS Research Ethics Review Board Approval (https://www.cdc.gov/nchs/nhanes/irba98.htm). Since all of the NHANES data are de-identified, the analysis of the data does not require Institutional Review Board approval (IRB) or further informed consent.
5.3 Study Population
Data of adults ≥ 40 years old in the NHANES database between 1999 and 2004 were extracted. The participants with incomplete data for ABI measures and other main study variables were excluded from the study cohort.
5.4 Assessment of ABI
ABI is the ratio of the blood pressure at the ankle to the blood pressure in the upper arm (brachium). It is usually regarded as an indicator for PAD in asymptomatic individuals. In the NHANES, the ABI exam was performed by trained health technicians in a specially equipped room in the mobile examination center (MEC). Participants lied supine on the exam table during the exam. Systolic pressure is measured on the right arm (brachial artery) and both ankles (posterior tibial arteries). Systolic blood pressure is measured twice at each site for participants aged 40–59 years and once at each site for participants aged 60 years and older. Participants are excluded from the exam if they have a bilateral amputation or weigh over 400 pounds (due to equipment limitations). Participants was categorized into two groups by ABI measures: abnormal group (ABI < 0.9) and normal group (ABI 0.9–1.3) for further comparison.
5.5 Assessment of Body Fluid Volume, Body Composition and Body Fat Distribution
In this NHANES database, body fluid measures including extracellular fluid volumes, intracellular fluid volumes, total water body volumes, and body fat including estimated fat mass, fat-free mass and percent body fat were determined by Bioelectrical impedance analysis (BIA). The NHANES bio-impedance spectroscopy (BIS) multi-frequency measurements were collected in the BIA examination. A small alternating current was passed through surface electrodes placed on the right hand and foot and the impedance to the current flow was measured by different electrodes placed adjacent to the injection electrodes. The voltage drop between electrodes provided a measure of impedance, or opposition to the flow of the electric current.
Data of body fat distribution in the present analysis were obtained from Dual-energy x-ray absorptiometry (DXA), which is the most widely accepted method of measuring body composition, due in part to its speed, ease of use, and low radiation exposure. The whole body DXA scans were administered in the NHANES MEC. In particular, DEXA scans were administered to eligible survey participants 8 years of age and older. Pregnant females, self-reported history of radiographic contrast material use in past 7 days, nuclear medicine studies in the past 3 days, and weight over 300 pounds or height over 6’5’’ were excluded from the examination. Total percent fat, percent fat of the limbs and truck were included in the analysis.
5.6 Demographic and Socioeconomic Status
The Family and Sample Person Demographics questionnaires were collected in the participants’ homes by trained interviewers using the Computer-Assisted Personal Interviewing (CAPI) system. Age, sex, and race/ethnicity were recorded using interviewer-administered questionnaires.
5.7 Laboratory Measurement
Blood specimens were collected at NHANES Mobile Examination Centers (MECs). Whole blood specimens were processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, and Centers for Disease Control and Prevention for analysis. Complete descriptions of the collection and analytical methods are available in the Laboratory data section of NHANES database. Individual’s laboratory data such as serum albumin, total bilirubin, hemoglobin level, C-reactive protein (CRP), homocysteine, folate and vitamin B12, level of total cholesterol and triglycerides, as well as white blood cell counts were identified and included in the analysis.
5.8 Statistical Analysis
To take complex sampling design of NHANES data into account, all analyses were performed using SAS survey analysis procedures to generate nationally representative estimates (SAS Institute Inc., Cary, NC, USA). Weighted mean and 95% confidence interval (CI) were presented for continuous variables; unweighted number and weighted proportion were presented for categorical variables. Since three cycles of data were combined in the current study, sample weights across survey cycles were constructed according to analytic guidelines published by National Center for Health Statistics.
Differences in means between groups of ankle brachial index (ABI) were compared using SURVEYREG procedure for continuous variables, while Rao-Scott chi-square test was performed to examine difference in the proportions between ABI groups using SURVEYFREQ procedure for categorical variables. Linear regression analysis and binary logistic regression analysis were performed to evaluate the association of ABI with body fluid and fat, as well as potential covariates such as socioeconomic status, biomarkers, comorbidity, behaviors, and intake of nutrients. Probabilities modeled are cumulated over the lower Ordered Values. Variables with p-value less than 0.05 in univariate analysis were considered as potential confounding factors. Multivariable models were then constructed by adding significant covariate pertaining to socioeconomic status, biomarkers/comorbidity/examination and behavior/nutrients intake sequentially. Each measure of body fluid and fat was performed in separate multivariable model. Since fat was measured by dual energy X-ray absorptiometry in which multiple imputation was performed to deal with missing data, all analyses in terms of fat distribution were performed separately by each of the five imputed datasets and then combined using MIANALYZE procedure to provide accurate estimates of standard error.