2.2. Body Composition Assessment
Volunteers, wearing form-fitting clothing without jewelry, came to the laboratory after consuming a light meal and emptying their bladders. Standing height and body weight were determined using standard medical equipment (SECA Stadiometer and SECA 762 scale; Hamburg, Germany).
Body composition estimation included BIA, SLSDI, and DXA administered in random order. Volunteers underwent whole-body BIA testing using a 50 kHz phase-sensitive impedance analyzer that introduced a sinusoidal, constant current (250 µA RMS) (BIVA, EFG 3 Monitor; Akern, Florence, Italy) using a tetrapolar surface electrode placement with paired current-injecting and voltage drop-measuring electrodes (BIAtrodes; Akern, Florence, Italy) separated a minimum of 5 cm and placed on the right wrist and ankle. Volunteers rested supine on a bed without contact with metal for 10 min. This BIA instrument provided direct measurements of resistance (R), reactance (Xc), and phase angle (PhA). The technical accuracy and precision of the BIA instrument were determined with a calibrated precision parallel circuit formed by a 384 Ω (±1%) resistor in parallel with a 780 pF (±2%) capacitor yielding 47 Ω of Xc at 50 kHz.
Body composition was estimated by using the following BI prediction models. Kyle et al. [
26] (BIA1):
FFM = 4.104 + 0.518 Ht2/R + 0.231 Wt + 0.130 Xc + 4.229 Sex* [1 for men and 0 for women]
Males: FFM = -9.88 + 0.65 Ht2/R + 0.26 Wt + 0.02 R
Females: FFM = -11.03 + 0.70 Ht2/R + 0.07 Wt + 0.02 R
Units are FFM (kg), height (Ht)2/R (cm2/Ω), weight (Wt, kg), and Xc (Ω).
We also estimated body composition using the proprietary equations of the BIA instrument manufacturer (Bodygram PLUS; Akern, Florence, Italy) (BIA3). This approach estimates the actual hydration (water content) of the FFM of an individual and uses the measured hydration in lieu of the customary 0.732 steady-state hydration ratio to estimate FFM (Akern Bodygram PRO; Florence, Italy). Fat mass is calculated as the difference between body weight and FFM.
A single SLSDI of each volunteer was obtained using the Fit.Your.Outfit (Pixelcando SL-Spain) smartphone APP implemented with a Cloud-based artificial intelligence (AI). An SLSDI of each volunteer was obtained without regard to background or illumination and transformed into an anonymous silhouette [
22]. The individual stood upright with the head positioned in the horizontal plane, arms fully extended alongside the body with feet and legs touching and aligned sagittal to the camera to provide a lateral profile of the body (
Figure 1). The smartphone cameras, either iOS- iPhone SE or iPhone 11 with CCD resolutions greater than 50 megapixels, were either pre-positioned on a stable tripod or held by a second individual who directed the handheld smartphone camera with the lens pointed at the middle of the standing height of each study participant. The AI system software automatically scaled all the digital pictures to a single homogeneous resolution of 5 megapixels and removed any background from the subject. The distance from the camera to the individual was 1.8 to 2.1 m with the feedback system of the smartphone APP. The operator downloaded and installed the Fit.Your.Outfit APP, available in iOS and Android from the APP stores, ensures the high technical quality of digital images for analysis to estimate body composition using proprietary software. The adequacy of the technical quality of the photograph is controlled and artifacts are prevented by using built-in sensors of modern smart devices and native libraries to detect specific anatomic nodal points (
Figure 1) to identify parallaxes, improper distance from an individual to a camera, and to recognize incorrect arm extension and position, improper alignment of legs and feet, and non-horizonal head position. The quality control protocol provides visual warning instructions to the operator to ensure acquisition of high-quality digital images for analysis.
Precision, determined as coefficients of determination and concordance coefficients for repeated profiles of SLSDI images and FM estimates were 0.996 and 0.997 (p < 0.0001), respectively, with no difference (<0.1 kg) between repeated images.
Reference whole-body composition was determined using DXA, a GE Lunar iDXAncore sn 200278 using software version 14.10.022 (Madison, WI). All participants were positioned supine and scanned within the dimensions of the DXA table. Precision estimates were 1.3% for FM and 0.5% for FFM.
Fluid distribution was evaluated using BIA measurements and bioelectrical impedance vector analysis (BIVA) [
28]. This method uses tetrapolar BIA and a 50 kHz phase-sensitive impedance device and provides whole-body, direct serial measurements of resistance (R), reactance (Xc), and phase angle (PhA). One measure was the ratio of ECW to total body water (TBW), ECW%, which was estimated with a proprietary model (Akern BodyGram PLUS; Florence, Italy). We also assessed fluid distribution (ECW/ICW) using PhA, which is inversely related to ECW/ICW [
29].
2.3. Statistical Methods
Statistical analyses were performed using SYSTAT version 13 (Systat Corporation; San Jose, CA, USA) and version 19.0.3 (MedCalc Software Bv, Ostend, Belgium). Descriptive data are expressed as mean ± SD. Statistical significance was set at p < 0.05.
Estimates of FM from the smartphone SLSDI and each BIA prediction model were compared with the reference DXA values. Measurement agreement was evaluated with concordance correlation coefficient (CCC) [
29], standard error of the estimate (SEE) between DXA and each method. Reference and estimates of body fat values in each sex group were compared separately with a paired t-test. Bland–Altman plot [
30,
31] was used to determine bias and limits of agreement (LOA) for the SLSDI and BIA methods compared to DXA determinations.
Effects of fluid distribution were evaluated in a sub-group of males with BMI greater than 25 kg/m2, the population indicator for overweight and obesity to contrast the effects of differences in adipose tissue with an unpaired t-test. Differences between group vector distributions were determined using Hotelling’s T2 test.