fractal and fractional

Article
Cole-Impedance Model Representations of Right-Side
Segmental Arm, Leg, and Full-Body Bioimpedances of Healthy
Adults: Comparison of Fractional-Order
Todd J. Freeborn * and Shelby Critcher

Department of Electrical and Computer Engineering, The University of Alabama, Tuscaloosa, AL 35487, USA;
[email protected]
* Correspondence: [email protected]

Abstract: The passive electrical properties of a biological tissue, referred to as the tissue bioimpedance,
are related to the underlying tissue physiology. These measurements are often well-represented
by a fractional-order equivalent circuit model, referred to as the Cole-impedance model. Objective:
Identify if there are differences in the fractional-order (α) of the Cole-impedance parameters that
represent the segmental right-body, right-arm, and right-leg of adult participants. Hypothesis: Cole-
impedance model parameters often associated with tissue geometry and fluid (R∞ , R1 , C) will be
different between body segments, but parameters often associated with tissue type (α) will not show
any statistical differences. Approach: A secondary analysis was applied to a dataset collected for an
agreement study between bioimpedance spectroscopy devices and dual-energy X-ray absoptiometry,
identifying the Cole-model parameters of the right-side body segments of N = 174 participants
using a particle swarm optimization approach. Statistical testing was applied to the different groups



of Cole-model parameters to evaluate group differences and correlations of parameters with tissue


features. Results: All Cole-impedance model parameters showed statistically significant differences
Citation: Cole-Impedance Model between body segments. Significance: The physiological or geometric features of biological tissues
Representations of Right-Side that are linked with the fractional-order (α) of data represented by the Cole-impedance model requires
Segmental Arm, Leg, and Full-Body further study to elucidate.
Bioimpedances of Healthy Adults:
Comparison of Fractional-Order.
Keywords: electrical impedance; bioimpedance; Cole impedance model; fractional-order equivalent
Fractal Fract. 2021, 5, 13.
circuit; fractional-order; segmental impedance; healthy adults
https://doi.org/10.3390/
fractalfract5010013

Academic Editor: Kurt Barbé

Received: 23 December 2020
1. Introduction
Accepted: 26 January 2021 The Cole-impedance expression, introduced by Kenneth Cole in 1940 [1], is an electri-
Published: 28 January 2021 cal impedance that has been widely utilized to represent the frequency-dependent electrical
impedance of biological tissues. Recently, this expression (or equivalent circuit model rep-
Publisher’s Note: MDPI stays neu- resentations of it) have been applied to model the frequency dependent impedance of
tral with regard to jurisdictional clai- human biceps tissues [2,3], skeletal muscle of mice [4], rabbit tissues [5], rat tissues [6],
ms in published maps and institutio- skin-electrode impedance [7], and modeling pork tissues during storage [8]. While these
nal affiliations. works are not an exhaustive summary of research that has employed the Cole-impedance
expression (or equivalent circuit models), this subset does highlight efforts that employ
this model to represent the electrical impedance of human and animal tissues. The original
Copyright: © 2021 by the authors. Li-
expression presented by Cole [1] is given by:
censee MDPI, Basel, Switzerland.
(r0 − r ∞ )
This article is an open access article z = z∞ + (1)
distributed under the terms and con-
1 + ( jωτ )α
ditions of the Creative Commons At-
where r0 and r∞ are described by Cole as the resistances at zero and infinite frequency, τ is
tribution (CC BY) license (https://
the time constant of the suspension and α a variable related to the membrane impedance
creativecommons.org/licenses/by/
(and that it varies as some power of the frequency) [1]. It is possible to represent (1) using
4.0/).

Fractal Fract. 2021, 5, 13. https://doi.org/10.3390/fractalfract5010013 https://www.mdpi.com/journal/fractalfract

Fractal Fract. 2021, 5, 13 2 of 15

the electrical equivalent circuit given in Figure 1a. This equivalent circuit will be referred to
as the Cole-impedance model throughout this manuscript and is composed of three circuit
elements (R∞ , R1 , and a constant phase element (CPE)). The impedance of this model is
given by:
R1
Z = R∞ + = R + jX (2)
1 + ( jω )α R1 C
where R and X represent the resistance and reactance (in Ohms) of the impedance, which
are often used to plot impedance datasets. Comparing the original impedance expression
from Cole given by (1) with the circuit model impedance given by (2), these expressions
1
are equivalent when: z∞ = r∞ = R∞ , r0 − r∞ = R1 , and τ = ( R1 C ) α . While resistors are
one of the traditional circuit elements commonly used in modeling impedance (in addition
to inductors and capacitors), the CPE is not. A CPE is a theoretical circuit element that has
the current-voltage characteristics given by:

dα v(t)
i (t) = C (3)
dtα
where 0 < α < 1 is the order of the CPE (and order of the fractional-order differentiation).
Beyond the Cole-impedance model, CPEs have been utilized in modeling the mechanical
impedance of lungs [9], the electrical impedance of lithium-ion batteries [10], and the
electrical impedance of concrete [11].
The current-voltage relationship given by (3) is a fractional-order differential equa-
tion. This places the CPE component into the realm of fractional calculus, the mathe-
matical field concerning integrals and derivatives of noninteger orders [12]. Concepts
from this field are being imported and investigated for modeling biological phenomena
as it offers methods to represent biological data using models with fewer parameters
than integer-order models [13,14]. For reference, the Grunwald–Letnikov definition of a
fractional derivative of order α is given by [15]:
∞
(−1)m Γ ( α + 1)
D α f ( x ) = lim ∑
h →0 m =0 h α m!Γ(α − m + 1)
f ( x − mh) (4)

where D α is the unified notation developed by Harold T. Davis to describe the order of
the differentiation or integration [16] (with further contemporary details available from
Ross [17]) and Γ(·) is the gamma function. Returning to the CPE, it has an electrical
impedance ZCPE = 1/C ( jω )α , where C and α are the pseudo-capacitance and fractional-
order, respectively. With the fractional-order fixed in the interval 0 < α < 1 (common for
biological tissues), the CPE has electrical characteristics that place it between a resistor
(α = 0) and an ideal capacitor (α = 1). It is for this reason that a CPE (with this limited band
of fractional-order) is also called a fractional-order capacitor. This reference to a fractional-
order capacitor is why it is represented using a capacitor symbol in Figure 1. The units of
a CPE/fractional-order capacitor proposed by Westerlund and Ekstam are F · secα−1 [18],
though often CPE units are presented in Farads. A simulation of (2) with parameters
R∞ = 100 Ω, R1 = 50 Ω, C = 10 µF · secα−1 , and α = 0.7, 0.85, 1 are given in the Nyquist
plot of Figure 1b. These specific values are selected because they are representative of values
reported for segmental tissues in this work (but this will be explored later sections). Notice
on an Nyquist plot, that this model has a semicircular behavior with the low and high
frequency reactance approaching 0 Ω and that the maximum reactance of the semicircle is
depressed for increasing values of α. Each of these simulations approaches 100 Ω (the R∞
value) at high frequencies which is why this component is referred to as the high-frequency
resistor. In addition, the simulations approach 150 Ω at low frequency (the R∞ + R1 value)
highlighting that both resistors are related to the low-frequency behavior.
Fractal Fract. 2021, 5, 13 3 of 15

30

1 25
α=1

sα C 20 α = 0.85

–Reactance (Ω)
α = 0.7
15

10

0 1 MHz R∞ = 100 Ω 10 Hz
R1 = 50 Ω
-5 C = 10 µF · secα−1
105 110 115 120 125 130 135 140 145
Resistance (Ω)
(a) (b)

Figure 1. (a) Electrical circuit representation of the Cole-impedance expression [1] and (b) MATLAB
simulated Nyquist plot of (2) with R∞ = 100 Ω, R1 = 50 Ω, C = 10 µF · secα−1 when α = 0.7, 0.85, 1.

Recently this model has been used to represent the impedance of localized biceps
tissues of healthy adults before/after exercise in the frequency band from 10 kHz to
100 kHz [2,3]. The aim of these efforts was to determine if the Cole-impedance model
parameters of localized tissues are altered as a result of exercise, which would support their
use as a reduced feature set for tissue monitoring over large datasets of raw impedances.
In their work, Freeborn and Fu reported statistically significant decreases in the resistance
parameters (R∞ , R1 ) but not the CPE parameters (C, α) comparing the pre-exercise and
post-exercise measurements [2]. In a further study of bicep tissue changes resulting from ec-
centric exercise, Fu and Freeborn reported statistically significant decreases in the resistance
parameters (R∞ , R1 ) and increases in C (but no changes in α) at timepoints 72 h and 96 h
after the eccentric exercise stimulus [3]. Of interest in both studies from a fractional-order
circuit model perspective, is that the fractional-order α did not show statistically significant
differences as a result of either exercise protocol and that localized biceps tissues (expected
to be composed primarily of skeletal muscle) had similar α (approximately 0.75). Similar
values were also reported by Regaud et al. for the CPE order of skeletal muscle from
sheep (0.71) [19]. Based on the similar values of α between these studies, Fu and Freeborn
hypothesized that the fractional-order may be linked to the tissue type (but this hypothesis
was not tested).
This provides the motivation for this work, to compare the Cole-impedance model
parameters that represent different segmental and full-body impedance measurements
of healthy adults. This study has a particular focus on the CPE parameters (C, α). It is
hypothesized that parameters linked to tissue geometry and fluid (R∞ , R1 , C) will be
different between body segments, but parameters linked to tissue type (α) will not show
any statistical differences. To test this hypothesis, a secondary analysis of measurements
collected/reported by Esco et al. in [20] was conducted. This dataset was collected during
the course of an agreement study between bioimpedance spectroscopy devices and dual-
energy X-ray absoptiometry for body composition determination [20] (but an investigation
of the Cole-model parameters was not within its scope). From this dataset, the Cole-
impedance parameters (R∞ , R1 , C, α) that represent the segmental tissue impedance of the
right-side full body, right arm, and right leg of 176 adult participants were identified using
a particle swarm optimization approach. These identified Cole-impedance parameters
were then analyzed using statistical tests to identify if there are significant differences
between parameters of different body segments. Finally, follow-up analyses to quantify
correlations between Cole-impedance parameters and dual-energy X-ray absorptiometry
(DXA) measures of soft tissues (tissue percent body fat) for these body segments were
completed. Based on these results, while R∞ and C parameters show strong correlations
Fractal Fract. 2021, 5, 13 4 of 15

with segmental lean tissue mass, the fractional-order (α) of the Cole-impedance parameters
does not show statistically significant correlations. The following sections of this work
detail the methods to collection the bioimpedance datasets, methods for their analysis and
comparison, the overall study results, and discussions of the results and their implications.

2. Materials and Methods

2.1. Study Participants
The data analyzed in this study was collected by the Exercise Physiology Lab research
group at the University of Alabama (UA), a subset of which was previously reported by
Esco et al. [20] for their agreement study between bioimpedance spectroscopy devices and
DXA for body composition determination. Complete details of the data collection process
are available in [20] but are also summarized here for easy reference.
During data collection, electrical impedance measurements and body composition
measurements were collected using a bioimpedance spetroscopy and a DXA device, respec-
tively. For this study, segmental measurements of the right arm, right leg, and right-side full
body of 185 participants (31.5 ± 14.8 years, 170.8 ± 8.9 cm, 76.6 ± 18.5 kg) were analyzed.
Measurements using both devices were collected from participants while in a supine body
position after having arrived at the Exercise Physiology Lab at the University of Alabama
in a fasted (8–12 h) euhydrated state. Additionally, participants were to have avoided
strenuous exercise in the day prior to testing. This study was reviewed and approved
by the institutional review board of The University of Alabama (15-019-ME). All study
participants reported no cardiovascular or metabolic disease and were not pregnant. Each
participant provided their written informed consent prior to data collection.

2.2. Dual-Energy X-ray Absorption Measurements

Dual-energy X-ray absorptiometry is a measurement method that utilizes the differ-
ences in absorption of X-ray photons at high and low energy to quantify bone and soft tissue.
Further technical details regarding this technique are available from Pietrobelli et al. [21]
for interested readers. DXA measurements of total tissue mass, fat tissue mass, and lean
tissue mass of each body segment (right arm, right leg, right-side full body) were collected
from each participant using a commercially available Lunar Prodigy Advance device from
GE Healthcare. This system provides the hardware and software necessary to collect body
composition assessments from a participant placed on the scanning bed. For this study,
whole body scans were completed while participants were on the scanning bed of the
device in a supine position with hands at their side and velcro straps wrapped at the ankles
and knees. After the whole-body scan, the DXA tissue measurements reported by the
device software were manually recorded for later analysis.

2.3. Electrical Impedance Measurements

The electrical impedance measurements of each body segment (right arm, right leg,
right-side full body) were collected from each participant using a tetrapolar electrode
configuration with an ImpediMed SFB7 device (shown in Figure 2a). The SFB7 is a commer-
cially available instrument marketed by ImpediMed for the estimation of body composition
in healthy individuals but is also widely utilized for clinical research [22,23]. This instru-
ment measures impedances at 256 discrete frequencies from 3 kHz to 1 MHz, with a
manufacturer rated accuracy of 1% (for impedances as low as 10 Ω); which has been
validated by Freeborn et al. [24]. The SFB7 uses a tetrapolar configuration of electrodes,
with two electrodes injecting the excitation current (I+, I−) and two electrodes measuring
the voltage response of the tissue segment (V+, V−). A tetrapolar configuration is uti-
lized to reduce (but cannot remove) the effects of the electrode/tissue interface impedance
which is typically much larger than the tissue impedance. For further details regarding
tetrapolar measurements, readers are recommended to review the works of Grimnes and
Martinsen [25] and Aliau-Bonet and Pallas-Areny [26].
Fractal Fract. 2021, 5, 13 5 of 15

The approximate locations of the electrodes for each body segment measured in this
study are detailed in Figure 2b. Electrodes were placed on the hand and shoulder, foot and
thigh, and hand and foot for the right arm, right leg, and right-side full body measurements,
respectively. For data collection in this study, adhesive Ag/AgCl electrodes were placed by
the study personnel on each of the body sites after cleaning with isopropyl alcohol and
removal of hair to improve contact with the skin. After electrode placement, participants
were asked to lay supine while their electrical impedance measurements were collected
using the SFB7 device interfaced to the electrodes. The cables of the SFB7 were manually
attached to the necessary electrode pairs for each measurement. Figure 2b illustrates the
connection of the SFB7 to the electrodes for the right-side full body measurement of a
participant. Measurements from each of the 3 body segments were saved on the instrument
for later download/post-processing in their raw format.

Electrode Locations

(a)

SFB7 V+

I+

V-

I-

(b)
Figure 2. (a) ImpediMed SFB7 device to collect participant electrical impedance using (b) referenced
electrode locations to measure segmental right arm, segmental right leg, and right full-body of each
study participant with tetrapolar configuration.

2.4. Outlier Identification/Removal

Before application of an optimization fitting procedure to identify the Cole-impedance
model parameters that best represented each individual impedance dataset (described
further in Section 2.5), the datasets were visually reviewed by the study team to identify
if they had features that would prevent their fitting. The features that were screened in
this step were negative reactances in the impedance plot at high frequency and motion
artifacts. Samples of these types of features in sample impedance datasets are given in
Figure 3b,c respectively. These artifacts are associated with errors in measurements and not
representative of the tissue. A total of 7 negative reactance cases (all present in right-leg
impedance datasets) and 2 cases of motion artifacts were identified. Note, because of the
statistical testing utilized later in this work (described in Section 3.1), all datasets from a
Fractal Fract. 2021, 5, 13 6 of 15

participant were excluded for those with a case of negative reactance or motion artifacts.
Therefore, a total of 9 participants datasets were removed from the analysis at this stage.
After parameter identification, the data was further analyzed for high-frequency hook
artifact or a poor fitting, both determined by visual inspection. Two datasets were identified
as having a hook artifact or poor fitting, shown in Figure 3a for reference. After this outlier
identification/removal, the datasets of N = 174 participants were used in the statistical
analysis. This process of outlier identification is summarized in Figure 4.

40 20 65
(a) (b) (c)
60
15
35 55

–Reactance (Ω)
–Reactance (Ω)
–Reactance (Ω)

10 50
30
45
5
40
25
0 35
20 30
-5
25
15 -10 Participant 149
20
240 250 260 270 280 290 300 310 320 330 280 285 290 295 300 305 310 315 320 325 330 360 380 400 420 440 460 480 500
Resistance (Ω) Resistance (Ω) Resistance (Ω)

Figure 3. Samples of participant data excluded (solid) from statistical analysis due to (a) high-frequency hook artifact,
(b) positive high-frequency reactance, and (c) motion artifacts. Simulations using identified Cole-impedance parameters
(dashed) shown to highlight poor fitting for datasets with these characteristics.
Collection

Data Collection
Full-body (n=185)
Right-arm (n=185)
Right-leg (n=185)

Outlier Criteria
Filtering

Data assessed for outliers 1. Negative Reactance

Full-body (n=0)
Right-Arm (n=0)
Right-Leg (n=7)

2. Motion Artifacts
Optimization

Parameter Extraction (PSO)

Full-body (n=176) Full-body (n=2)
Right-arm (n=176) Right-Arm (n=2)
Right-leg data (n=176) Right-Leg (n=2)

Exclusion Criteria
Eligibility

Data assessed for exclusion criteria 1. Hook artifact

Full-body (n=1)
Right-arm (n=0)
Right-leg (n=1)

2. Poor fitting
Data included in statistical analysis Full-body (n=0)
Included

Full-body (n=174) Right-arm (n=0)

Right-arm (n=174) Right-leg (n=0)
Right-leg (n=174)

Figure 4. Process applied to complete set of electrical impedance datasets for outlier identifica-
tion/removal, resulting in the use of N = 174 participant datasets (from an original set of N = 185).

2.5. Cole-Impedance Model Parameter Identification

For each collected bioimpedance measurement, the Cole-impedance model parameters
(R∞ , R1 , C and α) were extracted by applying a particle swarm optimization (PSO) to
identify the parameters that minimized the squared difference (of the real and imaginary
impedance components) between the experimental data (from 3 kHz to 200 kHz) and
the Cole impedance model. Metaheuristic optimization procedures, such as PSO, have
been previously utilized for estimating bioimpedance parameters from impedance datasets
showing accurate results [27,28]. Based on the accuracy of the PSO implementations
Fractal Fract. 2021, 5, 13 7 of 15

reported by Narango-Hernandez et al. for fitting bioimpedance data [28] and the ease of
implementation using available MATLAB functions, this approach was adopted in this
work. The objective function for the PSO optimization applied in this work is:
n
min f 0 ( x) =
x
∑ (Re{Zk (x) − yk })2 + (Im{Zk (x) − yk })2 (5)
k =1

where f 0 ( x ) is the objective function, n is the number of discrete frequencies used for
fitting, yk is the collected electrical impedance impedance at the k-th frequency, Zk ( x ) is the
impedance of the Cole impedance model with x (the vector of the model parameters R∞ ,
R1 , C, α), and Re and Im denote the real and imaginary components of the impedances.
This procedure was implemented using the particleswarm function available in MATLAB
with the following options: Swarm Size= 1000, Social Adjustment Weight = 1, MinNeigh-
borsFraction= 0.6, and the hybrid functionality was enabled to apply the fmincon solver
after the PSO solver terminated. Hybrid functions can obtain a more accurate solution
(to the PSO alone) by starting from the relatively rough solution found by the first solver.
This functionality is built into the MATLAB functions to implement the PSO and does
not require additional effort on the part of the user to setup. Constraints were also added
to
h the PSO implementation, with h and upper boundaries for [ R∞ i, R1 , C , α] fixed at
i lower
1 mΩ, 1 mΩ , 1 nF · secα−1 , 0.5 and 100 Ω, 100 Ω , 0.1 mF · secα−1 , 1.05 , respectively.

3. Results
A sample of participant impedances with simulations using the PSO identified Cole-
model parameters are given in Figure 5 for 6 different participants. In each of the subfigures,
the solid lines represent the experimental data and dashed lines represent the MATLAB
simulated Cole-impedance using (2). The line color is used to differentiate the body
segments with black, red, and blue corresponding to the right-side full-body, right-arm,
and right-leg data, respectively. Notice that each dataset, regardless of body segment has
an arc which is well-represented by the simulation using the Cole-impedance model (and
appropriate model parameters). To highlight the effect that outlier datasets have on the
PSO identification process, simulations using identified Cole-impedance parameters from
previously removed hook artifact data, positive reactance, and motion artifacts are shown
in Figure 3. In each case, the simulations shown as dashed lines show a significant deviation
compared to the experimental measurements (solid), indicating that the Cole-impedance
model is a poor fit when these artifacts are present.
To visualize the complete range of the set of Cole-impedance model parameters that
were identified from all participant datasets, histograms of R∞ , R1 , C, and α are detailed in
Figure 6. From these figures, there is significant overlap between the right arm and right
leg R∞ and R1 parameters and very little overlap between right arm/leg and right-side
full body parameters. The R∞ values range from approximately 100 Ω to 200 Ω for right
arm/leg and 300 Ω and 600 Ω for right side full-body. R1 ranges from approximately 50 Ω
to 125 Ω for right arm/leg and 150 Ω and 250 Ω for right side full-body. C is < 1 µF for
right leg/right side full-body and 1 µF to 6 µF for right arm. Of significant interest for
this study, is that the fractional-order (α) of the different body segments shows very little
overlap. With the right arm values ranging from approximately 0.6 to 0.73, full body values
ranging from 0.73 to 0.8, and right leg values ranging from 0.8 to 0.9. The median values of
the Cole-impedance model parameters from the complete set of extracted values from the
174 participants are given in Table 1.
Fractal Fract. 2021, 5, 13 8 of 15

80 80 80
Experimental Right Body
70 Fit Right Body 70 70
Experimental Right Arm
Fit Right Arm
60 Experimental Right Leg 60 60
Fit Right Leg

-Reactance (Ω)
-Reactance (Ω)

-Reactance (Ω)
50 50 50
40 40 40
30 30 30
20 20 20
10 10 10
Participant 1 Participant 61 Participant 90
0 0 0
150 200 250 300 350 400 450 500 550 600 650 200 300 400 500 600 700 800 900 200 300 400 500 600 700 800
Resistance (Ω) Resistance (Ω) Resistance (Ω)
50 70 70
45
60 60
40
50 50
-Reactance (Ω)

-Reactance (Ω)
35
-Reactance (Ω)

30 40 40
25 30 30
20
20 20
15
10 10 10
Participant 120 Participant 150 Participant 181
5 0 0
150 200 250 300 350 400 450 500 200 300 400 500 600 700 800 900 200 250 300 350 400 450 500 550 600 650 700
Resistance (Ω) Resistance (Ω) Resistance (Ω)

Figure 5. Samples of right-side full-body (black), right arm (blue), and right leg (red) bioimpedances collected from
participants (solid line) compared to simulations of Cole-impedance model using PSO identified parameters (dashed).

R∞ (Ω)

R1 (Ω)

C (µF · secα−1 )

Figure 6. Histograms of the Cole-impedance model parameters (R∞ , R1 , C, α) identified by the PSO to
best-fit the participant right-side full body (blue), right arm (orange), and right leg (yellow) datasets.
Fractal Fract. 2021, 5, 13 9 of 15

Table 1. Median Cole-impedance model parameters (R∞ , R1 , C, α) identified by the PSO to best-fit
the 174 participant right-side full body, right arm, and right leg bioimpedance datasets.

Body Segment R∞ (Ω) R1 (Ω) C µF · secα−1 α

Right-side Full Body 440.28 191.32 0.566 0.7428
Right Arm 191.39 92.51 2.57 0.6841
Right Leg 210.56 80.05 0.534 0.8374

3.1. Statistical Testing: Friedman Test

To test the study hypothesis (that the fractional-order, α, for each body segment
will be equal) statistical testing was utilized to compare the Cole-impedance impedance
parameters obtained from each body segment. Prior to the statistical comparison, the
normality of the parameters datasets were tested using the Shapiro–Wilk’s test (p < 0.05).
The assumption of normality for α for the full-body and right-arm segments were violated
but not for the right-leg. The assumption of normality for R∞ values were satisfied for the
right-leg dataset but not for the full-body and right-arm. The assumption of normality for
R1 values were satisfied for the full-body and right-leg but not the right-arm. Lastly, the
assumption of normality for C values were not satisfied for all body locations. Because some
groups within our dataset violated normality and were not independent, the nonparametric
Friedman test was utilized to determine if there were statistically significant differences
between the 3 related samples groups.
For the applied Friedman test (SPSS Statistics 26) statistical significance was accepted
at the p < 0.05 level. The medians for each set of parameters in this analysis are detailed
in Table 1. From the Friedman test, the fractional order (α) was statistically different for
all segments, χ2 (2) = 346.011, p < 0.005. From similar tests the R∞ , R1 , and C parameters
were statistically different for all segments (R∞ : χ2 (2) = 292.08, p < 0.005; R1 : χ2 (2) =
275.89, p < 0.005; C : χ2 (2) = 269.38, p < 0.005). Following each analysis, pairwise
comparisons were performed with a Bonferroni correction for multiple (3) comparisons.
For each parameter (R∞ , R1 , C, α) there were statistically significant differences (p < 0.0167)
for the right-arm/right-leg, right-arm/right-body, and right-leg/right-body comparisons.

3.2. Statistical Testing: Spearman Correlation

To determine if there were correlations (and their strengths) between the Cole-impedance
parameters and total tissue/tissue type, Spearman’s rank-order correlation coefficients
were calculated in MATLAB between each of the parameters (R∞ , R1 , C, α) for each body
segment and the DXA calcualted total tissue, lean tissue, and fat tissue (each in kg). The
scatterplots of these datasets are given in Figure 7. In each subplot, Figure 7a–d, there
are three scatterplots to visualize each of the Cole parameters with the segmental tissue
mass, segmental lean mass, and segmental fat mass for that body segment. Each body
segment is represented by a different color, with black/blue/red representing the right-side
full-body/right arm/right leg, respectively. Note that only 159 of the 174 participants with
identified Cole-impedance model parameters had available DXA data for this analysis.
The Spearman-rank correlation coefficients (rs ) and their corresponding statistical
significance (if p < 0.05) are presented in Table 2. Coefficients that were not statistically
significant are not reported. For the calculation of the statistical significance, a Bonferroni
correction was applied to compensate for the multiple correlations (4) per segmental
dataset. As an example of using Table 2, there was a statistically significant, moderate
negative correlation between total tissue mass of the right-side full-body and the R∞ Cole-
impedance parameter (p < 0.001, rs = −0.731), a very strong negative correlation between
lean tissue mass of the right-side full-body and R∞ (p < 0.001, rs = −0.862), and no
statistically significant correlation between fat tissue mass and R∞ .
Fractal Fract. 2021, 5, 13 10 of 15

Right-Side Full-Body Right Arm Right Leg

R∞ (Ω)

R1 (Ω)
500 200

100

0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Segmental Tissue (kg) Segmental Tissue (kg)

200

R1 (Ω)
500
R∞ (Ω)

100

0 0
0 10 20 30 40 50 60 70 80 0 0 10 20 30 40 50 60 70 80 0
Segmental Lean Tissue (kg) Segmental Lean Tissue (kg)
R∞ (Ω)

500 200

R1 (Ω)
100

0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
(a) Segmental Fat Tissue (kg) (b) Segmental Fat Tissue (kg)

1
C (µF · secα−1 )

5
0.8

0 0.6
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Segmental Tissue (kg) Segmental Tissue (kg)
C (µF · secα−1 )

1
5
0.8

0 0.6
0 10 20 30 40 50 60 70 80 0 0 5 10 15 20 25 30 35 40 45
Segmental Lean Tissue (kg) Segmental Lean Tissue (kg)
1
C (µF · secα−1 )

5
0.8

0 0.6
0 10 20 30 40 50 60 70 0 5 10 15 20 25 30 35
(c) Segmental Fat Tissue (kg)
(d) Segmental Fat Tissue (kg)

Figure 7. Scatterplots of (a) R∞ , (b) R1 , (c) C, and (d) α vs. total segmental tissue, lean tissue, and fat tissue for right-side
full body (black), right arm (blue), and right leg (red) impedance datasets.

Table 2. Spearman-rank correlation coefficients (rs ) and statistical significant (p) between Cole-impedance parameters and
total/lean/fat tissue masses for segmental measurements.

Total Tissue (kg) Lean Tissue (kg) Fat Tissue (kg)

Full-Body Arm Leg Full-Body Arm Leg Full-Body Arm Leg
R∞ (Ω)
rs (157) −0.731 −0.856 −0.641 −0.862 −0.903 −0.781 − − −
p <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 − − −
R1 (Ω)
rs (157) −0.375 −0.528 −0.222 −0.267 −0.517 − −0.323 − −0.307
p <0.001 <0.001 0.020 0.003 <0.001 − <0.001 − <0.001
C (µF · secα−1 )
rs (157) 0.682 0.636 0.542 0.612 0.545 0.611 0.334 0.366 −
p <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 −
α
rs (157) −0.384 − −0.334 −0.351 − −0.468 − −0.386 −
p <0.001 − <0.001 <0.001 − <0.001 − <0.001 −
Note: A blank cell (“−”) indicates no statistically significant correlation.
Fractal Fract. 2021, 5, 13 11 of 15

4. Discussion
The results of the statistical analysis support that R∞ , R1 , and C parameters of the
Cole-impedance model to represent the 3 body segments measured in this study are in
fact different. This aligns with the study hypothesis, that body segments with different
dimensions would have different R∞ , R1 , and C. The magnitude of differences also aligns
with expectations based on segmental dimensions. That is, the resistances (R∞ , R1 ) for the
right-side full-body measurements have a median value that is more than 2x the values
of the right arm and leg values (medians given in Table 1). The full-body measurements
capture tissues and fluids of the arm, leg, and torso which represents a greater resistive
path to the excitation current injected by the electrodes than the arm/leg alone and are
expected to be a major contribution towards these differences. This trend of increasing
resistance with increasing distance between electrodes also aligns with previous forearm
impedance measurements reported by Fu and Freeborn [29].
The moderate to strong correlations of R∞ with lean tissue mass in Table 2 aligns with
previous bioelectrical impedance studies to estimate skeletal muscle mass [30,31]. The
resistance measurement (either from a single frequency or the Cole-model estimated value)
is typically used as a variable in estimation equations for skeletal muscle and fat free mass.
As an example, Janssen et al. reported the following regression equation for skeletal muscle
mass (SM) in kg given by:
" #
Ht2
SM = × (0.401) + gender × (3.825) + age × (−0.071) + 5.102 (6)
R

where Ht is height (in cm), R is 50 kHz resistance (in Ω), gender is a binary variable
(men = 1, women = 0), and age is in years [30]. Notice in (6) that R is in the denominator of
the Ht2 /R term, which will reduce this terms contribution to the estimated skeletal muscle
mass for larger values of R. This is consistent with other reported regression equations to
estimate lean tissue mass [31]. The reported results in this work also align with this trend,
with lower resistance values being correlated with higher DXA estimated lean tissue mass.
The most significant result of this work is that the statistical comparison of the α values
in Figure 6 between segmental/full-body electrical impedance does not support the study
hypothesis. The initial hypothesis, that the value α would be strongly associated with
tissue type (skeletal muscle) and not have statistically significant differences between the
values identified for the different measured body segments of the study participants is not
supported. Based on the statistical comparison the median α for the right side full body,
arm, and leg were not equal, with values of 0.7428, 0.6841, and 0.8374, respectively. This
corresponds to differences of approximately −7.9% and 12.7% comparing the arm and leg
median α to the full-body value.
In terms of interpreting these results, it is important to analyze the underlying as-
sumptions of the initial study hypothesis, which are: (1) that segmental limb geometry
does not have a significant impact on α, and (2) segmental limb tissues were composed
primarily of skeletal muscle. The availability of DXA derived values of lean tissue mass, fat
mass, and bone mineral content for each participant provide the opportunity to explore the
tissue composition assumption. To visualize tissue composition with DXA measurements
the lean tissue, fat mass, and bone mineral content (BMC) as a percentage of the total
body mass (for the right side body, right arm, and right leg) was generated and plotted in
Figure 8. Each vertical bar represents a single participant, with orange, red, and blue used
to represent the percentage to segmental/body composition of BMC, fat tissue, and lean
tissue, respectively. The mean (±SD) contribution of lean tissue from right arm, leg, and
body measurements are 69.5% ± 9.4%, 64.5% ± 8.3%, and 66.9% ± 8.4%. This supports that
full body and segmental tissues are composed primarily of lean tissues/skeletal muscle.
As a result, it is likely that the segmental limb geometry does in fact have a significant
impact on α.
Fractal Fract. 2021, 5, 13 12 of 15

BMC Fat Tissue Lean Tissue

Composition (%) Composition (%) Composition (%)

100

Right Arm
50

0
100
Right Leg

50

0
100
Right Body

50

0
Participant
Figure 8. DXA derived contributions of bone mineral content (orange), fat tissue (red), and lean
tissue (blue) to overall composition of right arm, right leg, and right body of study participants.

To further explore recent reports of the fractional-order α of Cole-parameters estimated

from localized or segmental tissues of humans and animals (and compare them to the
results of this work), a summary of recent studies is provided in Table 3. This table details
the animal/human population from which measurements were collected, the muscle
location (if available), the measurement approach (Ex Vivo or surface), frequency band of
measurement, and the range of α that was reported. While the range of fractional-orders are
all in the approximate range of 0.52 < α < 0.97, highlighting that a fractional-order model
has provided good fit of experimental bioimpedance data, there is a significant overlap
between α across the studies without any tight groupings for specific test conditions. For
example, measurements of excised skeletal muscle tissue and exposed skeletal muscle
tissue in animals have overlapping α compared to segmental surface measurements in
animal/human populations. However, it is important to note that the measurements
summarized from these studies all have significantly different setups and test populations;
which includes different electrode spacing, tissue contributions, tissue geometry, sample
populations, and underlying physiology. So while reports from literature support that a
wide range of α values has been determined from lean tissues, it does not provide a clear
answer regarding what features α may be linked.
While these results do not support the initial study hypothesis, they do highlight
that continued research is needed to elucidate what physiological or geometric features of
biological tissues are strongly linked with the fractional-order (α) of data represented by the
Cole-impedance model. Even though the Cole-impedance model continues to provide a
good “fit” with experimental data, the potential insights that it can offer regarding skeletal
muscle cannot be unlocked until this relationship is understood in greater detail. In fact, this
supports previous concerns of McAdams and Jossinet, who have reported limitations and
sources of error in using equivalent circuit modeling to represent the electrical properties
of biological tissues [37], which they recommend only after extensive study of the system
under different conditions. This recommendation should be adopted in future studies to
investigate how α is modified in biological tissues under different conditions to further
understand what conditions it is most strongly associated.
Fractal Fract. 2021, 5, 13 13 of 15

Table 3. Summary of fractional-order (α) reported from studies of skeletal muscle tissue impedance utilizing Cole-impedance model.

Authors Sample Population Muscle Location Frequency Band Fractional-Order (α)

Longitudinal:
Gemellus
Rigaud et al. [19] Sheep 7.2 kHz–720 kHz 0.73 ± 0.05
(Ex Vivo)
Traverse: 0.78 ± 0.02
Longitudinal (Young):
0.682
Gastrocnemius Traverse (Young): 0.660
Arnold et al. [32] Mice 1 kHz–10 MHz
(Surface) Longitudinal (Aged):
0.794
Traverse (Aged): 0.656
Longitudinal:
Gastrocnemius
Nagy et al. [33] Mice 1 kHz–10 MHz 0.709–0.760
(Surface)
Traverse: 0.713–0.748
Longitudinal:
Gastrocnemius
Clark-Matott et al. [34] Mice 1 kHz–10 MHz 0.522–0.677
Ex Vivo
Traverse: 0.690–0.784
Longitudinal:
Gastrocnemius 0.528 ± 0.012
Sanchez, Bragos, & (Ex Vivo) 1 kHz–1 MHz Traverse: 0.729 ± 0.01
Rat
Rutkove [35] Soleus 1 kHz–1 MHz Longitudinal:
(Ex Vivo) 0.695 ± 0.02
Traverse: 0.736 ± 0.01
Pre Exercise:
Healthy Biceps 0.552–0.781
Freeborn & Fu [2] 10 kHz–100 kHz
Adults (Surface) Post Exercise:
0.552–0.779
Exercised: 0.621–0.745
Healthy Biceps
Fu & Freeborn [3] 10 kHz–100 kHz Unexercised:
Adults (Surface)
0.628–0.766
Healthy
Sato et al. [36] Men Lower Extremities 5 kHz–250 kHz 0.71 ± 0.03
(Surface)
Right-Body:
Right-Body, Segmental 0.687–0.769
This work Healthy Adults 3 kHz–200
Arm/Leg Right Arm: 0.606–0.725
Right Leg: 0.778–0.971

5. Conclusions
While the Cole-impedance model is a fractional-order circuit model that well repre-
sents the experimental impedance measurements of segmental body impedances (right-side
full-body, right arm, and right leg), the fractional-order (α) parameter of this model showed
statistically significant differences comparing the segmental measurements of healthy
adults reported in this study. This supports that the fractional-order (α) is not strongly as-
sociated with tissue type (i.e., skeletal muscle) which warrants further research to elucidate
the tissue features to which it is most strongly associated towards advancing understanding
of this model and tissue bioimpedance.

Author Contributions: Conceptualization, T.J.F.; methodology, T.F. and S.C.; formal analysis, T.J.F.
and S.C.; data organization, T.J.F. and S.C.; data visualizations, T.J.F. and S.C.; writing—original draft,
review, and editing, T.J.F. and S.C. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Fractal Fract. 2021, 5, 13 14 of 15

Data Availability Statement: Data is contained within the article.

Acknowledgments: The authors would like to thank Michael Esco and the faculty/students/staff of
the Exercise Physiology Lab at the University of Alabama for providing access to the datasets they
had previously collected for the analyses in this study.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Cole, K. Permeability and impermeability of cell membranes for ions. Cold Spring Harbor Symp. Quant. Biol. 1940, 8, 110–122.
[CrossRef]
2. Freeborn, T.J.; Fu, B. Fatigue-induced cole electrical impedance model changes of biceps tissue bioimpedance. Fractal Fract. 2018,
2, 27. [CrossRef]
3. Fu, B.; Freeborn, T. Cole-impedance parameters representing biceps tissue bioimpednace in healthy adults and their alterations
following eccentric exercise. J. Adv. Res. 2020, 25, 285–293. [CrossRef] [PubMed]
4. Sanchez, B.; Li, J.; Geisbush, T.; Bardia, R.; Rutkove, S. Impedance alterations in healthy and diseased mice during electrically
induced muscle contraction. IEEE Trans. Biomed. Eng. 2016, 63, 1602–1612. [CrossRef]
5. Al-Surkhi, O.; Naser, R. Detection of cell morphological changes of ischemic rabbit liver tissue using bioimepdance spectroscopy.
IEEE Trans. Nanobiosci. 2018, 17, 402–408. [CrossRef]
6. Parramon, D.; Erill, I.; Guimer, A.; Ivorra, A.; Munoz, A.; Sola, A.; Fondevila, C.; Garcia-Valdecasas, J.; Villa, R. In vivo detection
of liver steatosis in rats based on impedance spectroscopy. Physiol. Meas. 2007, 28, 813–828. [CrossRef]
7. Taji, B.; Chan, A.; Shirmohammadi, S. Effect of Pressure on Skin-Electrode Impedance in Wearable Biomedical Measurement
Devices. IEEE Trans. Instrumen. Meas. 2018, 67, 1900–1912. [CrossRef]
8. Bai, X.; Hou, J.; Wang, L.; Wang, M.; Wang, X.; Wu, C.; Yu, L.; Yang, J.; Leng, Y.; Sun, Y. Electrical impedance analysis of pork
tissues during storage. Food Meas. 2018, 12, 164–172. [CrossRef]
9. Ribeiro, D.; Abrantes, J. Application of electrochemical impedance spectroscopy (EIS) to monitor the corrosion of reinforced
concrete: A new approach. Constr. Build. Mater. 2016, 111, 98–104. [CrossRef]
10. Westerhoff, U.; Kurbach, K.; Lienesch, F.; Kurrat, M. Analysis of Lithium-Ion Battery Models Based on Electrochemical Impedance
Spectroscopy. Energy Technol. 2016, 4, 1620–1630. [CrossRef]
11. Ionescu, C.; Tenreiro Machado, J.; De Keyser, R. Modeling of the Lung Impedance Using a Fractional-Order Ladder Network
With Constant Phase Elements. IEEE Trans. Biomed. Circuits Syst. 2011, 5, 83–89. [CrossRef] [PubMed]
12. Podlubny, I. Fractional Differential Equations; Academic Press: Cambridge, MA, USA, 1998.
13. Freeborn, T.J. A survey of fractional-order circuit models for biology and biomedicine. IEEE J. Emerg. Sel. Top. Circuits Syst. 2013,
3, 416–424. [CrossRef]
14. Ionescu, C.; Lopes, A.; Copot, D.; Machado, J.T.; Bates, J. The role of fractional calculus in modeling biological phenomena: A
review. Commun. Nonlinear Sci. Numer. Simul. 2017, 51, 141–159. [CrossRef]
15. Shantanu, D. Functional Fractional Calculus for System Identification and Controls; Springer: Berlin/Heidelberg, Germany, 2008.
16. Davis, H.T. The Theory of Linear Operators; Principia Press: Grand Rapids, MI, USA, 1936.
17. Ross, B. Fractional Calculus. Math. Mag. 1977, 50, 115–122. [CrossRef]
18. Westerlund, S.; Ekstam, L. Capacitor theory. IEEE Trans. Dielectr. Elec. Insul. 1994, 1, 586–839. [CrossRef]
19. Rigaud, B.; Hamzaoui, L.; Frikha, M.R.; Chauveau, N.; Morucci, J.P. In vitro tissue characterization and modelling using electrical
impedance measurements in the 100 Hz–10 MHz frequency range. Physiol. Meas. 1995, 16, A15–A28. [CrossRef]
20. Esco, M.R.; Fedewa, M.V.; Freeborn, T.J.; Moon, J.R.; Wingo, J.E.; Cicone, Z.; Holmes, C.J.; Hornikel, B.; Welborn, B. Agreement
between supine and standing bioimpedance spectroscopy devices and dual-energy X-ray absorptiometry for body composition
determination. Clin. Physiol. Funct. Imaging 2019, 39, 355–361. [CrossRef]
21. Pietrobelli, A.; Formica, C.; Wang, Z.; Heymsfield, S. Dual-energy X-ray absorptiometry body composition model: review of
physical concepts. Am. J. Physiol. Endocrinol. Metab. 1996, 271, E941–E951. [CrossRef]
22. Pateyjohns, I.; Brinkworth, G.; Buckley, J.; Boakes, M.; Clifton, P. Comparison of three bioelectrical impedance methods with DXA
in overweight and obese men. Obesity 2006, 14, 2064–2070. [CrossRef]
23. Vine, S.; Painter, P.; Kuskowski, M.; Earthman, C. Bioimpedance spectroscopy for the estimation of fat-free mass in end-stage
renal disease. e-SPEN Eur. J. Clin. Nutr. Metab. 2011, 6, e1–e6. [CrossRef]
24. Freeborn, T.J.; Milligan, A.; Esco, M.R. Evaluation of ImpediMed SFB7 BIS device for low-impedance measurements. Measurement
2018, 129, 20–30. [CrossRef]
25. Grimnes, S.; Martinsen, O. Journal of Physics D: Applied Physics Sources of error in tetrapolar impedance measurements on
biomaterials and other ionic conductors. J. Phys. D Appl. Phys. 2006, 20, 9–14.
26. Aliau-Bonet, C.; Pallas-Areny, R. On the Effect of Body Capacitance to Ground in Tetrapolar Bioimpedance Measurements.
IEEE Trans. Biomed. Eng. 2012, 59, 3405–3411. [CrossRef] [PubMed]
27. Yousri, D.; AbdelAty, A.; Said, L.; AboBakr, A.; Radwan, A. Biological inspired optimization algorithms for cole-impedance
parameters identification. Int. J. Electron. Commun. 2017, 78, 79–89. [CrossRef]
Fractal Fract. 2021, 5, 13 15 of 15

28. Naranjo-Hernandez, D.; Reina-Tosina, J.; Roa, L.; Barbarov-Rostan, G.; Areste-Fosalba, N.; Lara-Ruiz, A.; Cejudo-Ramos, P.;
Ortega-Ruiz, F. Smart bioimpedance spectroscopy device for body composition estimation. Sensors 2020, 20, 70. [CrossRef]
29. Fu, B.; Freeborn, T. Estimating localized bio-impedance with measures from multiple redundant electrode configurations.
In Proceedings of the 2018 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society,
Honolulu, HI, USA, 17–21 July 2018; pp. 4351–4354.
30. Janssen, I.; Hymsfield, S.; Baumgartner, R.; Ross, R. Estimation of skeletal muscle mass by bioelectrical impedance analysis.
J. Appl. Physiol. 2000, 89, 465–471. [CrossRef]
31. Kyle, U.; Bosaeus, I.; De Lorenzo, A.; Deurenberg, P.; Elia, M.; Gomez, J.; Heitmann, B.; Kent-Smith, L.; Melchior, J.; Pirlich, M.;
et al. Bioelectrical impedance analysis—Part i: Review of principles and methods. Clin. Nutr. 2004, 23, 1226–1243. [CrossRef]
32. Arnold, W.; Taylor, R.; Li, J.; Nagy, J.; Sanchez, B.; Rutkove, S. Electrical impedance myography detects age-related muscle change
in mice. PLoS ONE 2017, 12, e0185614. [CrossRef]
33. Nagy, J.; Kapur, K.; Taylor, R.; Sanchez, B.; Rutkove, S. Electrical impedance myography as a biomarker of myostatin inhibition
with ActRIIB-mFc: a study in wild-type mice. Future Sci. OA 2018, 4, FSO308. [CrossRef]
34. Clark-Matott, J.; Nagy, J.; Sanchez, B.; Taylor, R.; Riveros, D.; Abraham, N.; Simon, D.; Rutkove, S. Altered muscle electrical tissue
properties in a mouse model of premature aging. Muscle Nerve 2019, 60, 801–810. [CrossRef]
35. Sanchez, B.; Bragos, R.; Rutkove, S. Differentiation of the intracellular structure of slow- versus fast-twitch muscle fibers through
evaluation of the dielectric properties of tissue. Phys. Med. Biol. 2014, 59, 2369–2380. [CrossRef] [PubMed]
36. Sato, H.; Nakamura, T.; Kusuhara, T.; Kenichi, K.; Kuniyasu, K.; Kawashima, T.; Hanayama, K. Effectiveness of impedance
parameters for muscle quality evaluation in healthy men. J. Physiol. Sci. 2020, 70, 53. [CrossRef] [PubMed]
37. McAdams, E.; Jossinet, J. Problems in equivalent circuit modelling of the electrical properties of biological tissues. Bioelec-
trochem. Bioenerg. 1996, 40, 147–152. [CrossRef]