Relationship of Phase Angle, Echo Intensity, and Muscle Thickness with Isokinetic Knee Extensor Strength and Associated Motor Functions in Young Adults

Background: There is no comprehensive report on quadriceps femoris muscle parameters’ association with isokinetic knee extensor strength, and no study has comprehensively claried the relationship with motor function. To investigate the relation of isokinetic knee extensor strength and motor functions with phase angle, muscle echo intensity, and muscle thickness, to nd alternative methods for knee extensor strength assessment. Methods: This cross-sectional study evaluated the associations between phase angle (PA) (measured using bioelectrical impedance analysis), muscle echo intensity (EI), and muscle thickness (MT) (measured using ultrasound) and isokinetic knee extensor strength (measured as maximum torque at 60 degree/s using an isokinetic dynamometer), and motor functions evaluated by gait speed (usual and maximum), ve chair stands time, and stand up score. Results: The study comprised of 33 young healthy adults (12 males and 21 females; mean age, 22.2 years). Both sexes showed a signicant correlation between isokinetic knee extensor strength and PA (males, r = 0.65, females, r = 0.54), muscle EI (males, r = -0.53, females, r = -0.54), and MT (males, r = 0.34, females, r = 0.38). In addition, a signicant correlation was found between PA and maximum gait speed (males, r = 0.64, females, r = 0.44), ve chair stands time (males, r = -0.69, females, r = -0.62), and stand up score (females, r = 0.45), and between muscle EI and maximum gait speed (males, r = -0.34, females, r = -0.42), ve chair stands time (males, r = 0.37, females, r = 0.33), and stand up score (females, r = -0.30). Conclusions: The study suggests the potential of phase angle and muscle echo intensity as alternative assessment methods for isokinetic knee extensor strength.


Background
Generally, knee extension muscle strength is assessed using isokinetic muscle strength measurement devices, and is used as a measurable item in the decision criteria for returning to sports [1]. The advantage of evaluating muscle strength using an isokinetic muscle strength measurement device is that it provides resistance that is almost equal to the force output by the participants, so the participant does not receive unreasonable resistance [2], making it possible to measure muscle strength safely and accurately. However, isokinetic muscle force measurement devices are very expensive and have limited application in terms of the measurement location. In recent years, there have been reports of phase angle (PA), estimated using bioelectrical impedance analysis measurements [3], and muscle echo intensity (EI) and muscle thickness (MT), estimated using ultrasound [4], being related to knee extension muscle strength. However, there are no reports that examined them as knee extensor strength evaluation methods or comprehensively clari ed their relationship with motor functions in healthy young adults.
The PA is an index that is directly calculated from the measured values by the bioelectrical impedance analysis (BIA) [5] without using estimating equations, and is considered to re ect the physiological function at cellular level [6,7]. The BIA method measures the resistive and capacitive components of a biological tissue's ability to interfere with alternating currents. PA is the difference between the overall current and voltage, and indicates the structural integrity (quality) of the cell [8,9]. PA is reported to correlate with isometric knee extensor strength, maximal walking speed, and ve-repetition chair stand time in stable chronic obstructive pulmonary disease outpatients [3]. Therefore, PA is expected to play a role as a comprehensive indicator of physical characteristics and motor function. However, the relationship between isokinetic knee extensor strength, PA, and motor function is unclear. Muscle EI is an established method for evaluating muscle quality using diagnostic ultrasound. Muscle EI is quanti ed using gray-scale ultrasound, which entails the analysis of 256 grayscale levels from 0 to 255 and correlates with the amount of non-contractile tissue in skeletal muscle [4,10]. This non-invasive and safe method allows easy assessment of the proportion of non-contractile intramuscular elements, with an increase in EI being primarily interpreted as increased amounts of fat and connective tissue [10][11][12]. A predominantly white appearance suggests that there is a substantial amount of non-contractile tissue in the skeletal muscle, whereas a black appearance suggests that there is little non-contractile tissue [10].
The validity of ultrasound measurements of muscle ber mass and non-contractile tissues has been proven in comparison studies using magnetic resonance imaging [13,14]. Previous studies have reported associations between isokinetic knee extensor strength and quadriceps muscle EI in healthy young adults [15], and between muscle echo intensity and usual walking speed in the elderly [16]; however, the associations with other motor functions remain unclear. MT is used as an index of muscle mass.
Generally, MT is measured by transverse imaging with a probe in perpendicular contact with the muscle ber. The validity of MT has been demonstrated by its high correlation with actual measurements using cadavers [17,18] and MRI imaging [19,20]. The relationship between isokinetic knee extensor strength and quadriceps MT, and MT and normal walking speed has been shown in the elderly [4], but the relationship between MT and motor function is unclear.
There is no comprehensive report on quadriceps femoris muscle parameters' association with isokinetic knee extensor strength, which is used as one of the criteria for returning to sports, and no study has comprehensively clari ed the relationship with motor function. If the relationship between PA, muscle echo intensity, MT obtained from ultrasound, and motor function can be clari ed, it will be possible to evaluate muscle strength more easily and plan muscle strengthening programs from a more multifaceted perspective. We hypothesized that PA and muscle echo intensity, which are used for qualitative assessment of muscle, were more relevant to motor functions and isokinetic strength than MT, which is used as an index of muscle mass. Therefore, the purpose of this study was to clarify the relationship between the isokinetic knee extensor strength (and the related alternative methods of its evaluation methods) and motor functions in young healthy adults. The results are expected to make it possible to evaluate muscle strength more easily and from multiple functional perspectives.

Study Design and Participants
This was a cross-sectional study. We recruited healthy young adults from universities and graduate schools to participate in the study through university campus posters. The inclusion criteria were healthy young adult volunteers, 18-35 year old, who had not engaged in any regular and systematic training programs in the previous 6 months. The exclusion criteria were as follows: inability to walk without assistive devices and reported history of lower limb trauma or surgery, neuromuscular disorder, or acute or chronic diseases that might have impaired their muscle strength and power; any history of metabolic, hormonal, and cardiovascular diseases; and those taking any medication with an in uence on hormonal or neuromuscular metabolism. Participants were informed about the design of the study with speci c information on the possible risks and any discomfort related to the procedures that could occur. All participants completed and signed an informed consent form. All study procedures were conducted in accordance with the 1964 Declaration of Helsinki and its later amendments, and the study was approved by the Ethics Committee of Tokyo Metropolitan University Graduate School and Human Health Sciences (No. 20001).

Measurement of Study Parameters
Phase Angle Body composition parameters, including PA, were obtained using a multi-frequency bioelectrical impedance analyzer (Msd-100, TANITA Corp, Tokyo, Japan) and the 4-electrode method. The current applied was 100 µA or less, and the measurement frequencies were 5, 10, 20, 25, 40, 50, 100, 200, 250, and 500 kHz. The current values were in accordance with the standards established by the International Electrotechnical Commission. Disposable electrocardiogram electrodes (RedDot 2330; 3M Japan Ltd., Tokyo, Japan) were used to apply the current by placing them at the centers of the insteps of both feet. The detecting electrode was a belt-type stainless steel plate, 1 cm wide, rmly placed, and then looped around the measurement location, as shown in Fig. 1. The thigh and the stainless-steel plate of the electrode were wiped with a damp cloth, and the electrode was brought into contact with the sites 7 cm proximal and 7 cm distal to the midpoint connecting the greater trochanter to the lateral condyle of the femur. The measurement was performed in the supine position, and the right thigh was measured. The participants held their arms and legs to avoid contact with any other body segments during the procedure. According to the recommendations for clinical application of BIA [5], PA at 50 kHz was used for the analysis. The PA was calculated using the equation: based on Xc (reactance) and R (resistance).
For all measurements, BIA measurements were performed rst. To assess test-retest reliability, intra-class correlation coe cients (ICC (1.1)) were evaluated in six male and six female participants with a mean age of 21.5 ± 0.7 and 21.6 ± 0.7 years, respectively. The ICC (1.1) values of PA were 0.97 for both males and females.

Muscle Thickness and Echo Intensity
MT and muscle EI assessments were performed according to a previous study [21,15]. ) of the right leg were obtained using a B-mode ultrasound imaging device (Aplio i800; Canon Medical Systems Inc., Tokyo, Japan) and a multi-frequency linear probe (i18LX5; 18 MHz; probe width, 64 mm). All ultrasound images were obtained using the following acquisition parameters: gain, 80 dB; depth, 40 mm. All measurements were performed under the same conditions. Before the measurement, the participants rested in a supine position with their lower limbs relaxed and fully extended for 15 min to allow uid shifts to stabilize [22]. The probe was positioned perpendicular to the longitudinal axis of the quadriceps femoris muscle. A water-soluble gel was used to provide acoustic contact, and care was taken not to compress the subcutaneous tissue. The probe was xed at the position where the femur was the whitest and clearest, as depicted on the ultrasound monitor and just below its center. The images of the MT were obtained as the sum of the thicknesses of the RF and VI at the midpoint between the lateral condyle of the femur and the greater trochanter. The EI images were obtained as the muscle EI for the VM at the distal third of a line drawn from the lateral condyle of the femur to the greater trochanter. The ultrasound images were stored in an ultrasound machine for future analyses. Medical image processing, analysis, and visualization software (version 1.48; National Institutes of Health, Bethesda, MD, USA), was used to analyze the images on a personal computer. A rectangular region of interest (ROI), including as much muscle as possible but avoiding the bone and surrounding fascia, was determined for the EI calculation of the muscle. The mean EI of each muscle was determined using a standard histogram gray-scale function and expressed as a value between 0 (black) and 255 (white). Same investigator performed all the calculations. To assess test-retest reliability, intraclass correlation coe cients (ICC(1.1)) were evaluated in six female participants with a mean age of 21.5 ± 0.7 years. The ICC (1.1) values of MT and EI were 0.90 and 0.85, respectively.

Maximum Knee Extension Strength
The maximum knee extension strength was assessed according to a previous study [15]. The maximal isokinetic strength of the knee extensors on the right side was measured using an isokinetic dynamometer (Cybex Norm; CSMi, Computer Sports Medicine Inc., Stoughton, MA, USA). The participants were seated with a hip exion of 85 ° (0 ° being the anatomic position) and the lateral condyle of the right leg aligned with the axis of rotation of the dynamometer. After two practice trials, isokinetic strength (N) was measured for three repetitions, submaximal isokinetic knee extension/ exion repetitions at a velocity of 60°/s; the maximum value was then used. The torque (Nm) was calculated by multiplying the strength (N) by the lever arm (m).

Motor Functions
Gait speed assessment was performed according to a previous study [23]. Gait speed was measured in seconds using a stopwatch. The participants were asked to walk on a at and straight surface. The usual and maximum walking speeds were then measured. Usual meant walking at usual speed and the maximum was as fast as possible, and the measurement was performed twice to calculate the average value. Two markers were used to indicate the start and end of a 5 m walk path, with a 3 m section to be traversed before passing the start marker, so that participants were already walking by the time they reached the timed path. Participants were asked to continue walking for an additional 3 m past the end of the path to ensure a consistent walking pace while on the timed path. The time measurement started when the trunk crossed the start line of the measurement section and ended when the trunk crossed the goal line.
The ve-repetition chair stand test was performed according to a previous study [24]. Measurements were taken using a platform with a seat height of 40 cm from the oor without a backrest and armrests. Seated participants were asked to move forward on the chair seat until the feet were at on the oor and to fold their upper limbs across the chest. Participants were then instructed to stand up all the way and sit down once without using their upper limbs. For those unable to complete the initial maneuver or who required assistance, the test was terminated. If successful on the initial sit to stand, participants were then asked to stand up all the way and sit down landing rmly, as fast as possible, ve times without using the arms. Timing with a stopwatch was started when the buttocks got out of the seat and stopped at the fth upright posture; the time taken was recorded as the participant's score.
The stand-up test was performed according to a previous study [25]. This test assesses the participants' leg strength by having them stand up, using one or both legs, from a speci ed height, and maintaining their posture. Participants were requested to stand from four different height platforms (10, 20, 30, and 40 cm) with one or both legs. If a subject succeeded in standing up and maintaining that posture for 3 s, the trial was judged as complete. Participants were allocated a score of 0-8 based on their performance, and a score of 0 was given when the participants were unable to stand from a platform of 40 cm with both legs, and a score of 8 was given when it was able to stand from a platform of 10 cm with one leg. Higher scores indicated a better ability to stand up.

Statistical Analysis
The Shapiro-Wilk normality test was used to verify the distribution of data. The stand-up score is reported as median (25, 75 percentile), and all other values are reported as mean ± standard deviation (SD). The Pearson product-moment correlation test was used to investigate possible associations between the parametric parameters analyzed. For nonparametric data, the Spearman rank correlation test was used. Statistical signi cance was de ned as a p-value < 0.05. Since males are larger than females, for the PA, muscle EI, MT, and maximum force during isokinetic knee extension at 60°/s, all analyses were performed by sex. Statistical analysis was performed using SPSS Statistics for Windows, version 26.0, (IBM Corp., Armonk, NY, USA).

Results
The study participants included 33 participants (mean age ± SD, 22.2 ± 2.2 years; range, 20-32), including 12 males and 21 females. Their baseline physical characteristics and study parameters measurements are shown in Table 1. The correlation coe cients between PA, muscle EI, MT, and motor function are summarized in Table 2  The stand-up score is represented as median (25,75 percentile). Other values are presented as mean ± SD. a.u.: arbitrary unit Table 2 Correlation coe cient between phase angle, muscle echo intensity, muscle thickness and motor functions Males / Females, a.u.: arbitrary unit, *p < 0.05, **p < 0.01, statistical difference.

Discussion
In this study of 33 healthy young adults, both sexes showed a signi cant correlation between maximum torque during isokinetic knee extensor strength and PA, muscle EI, and MT. In addition, a signi cant correlation was found between PA as well as muscle EI and maximum gait speed, ve chair stands times, and stand-up scores. In contrast, MT did not correlate with any motor function.
While the correlation coe cients was highest between PA and knee extensor strength, it was lowest between MT and knee extensor strength. No previous studies were found for the relationship between PA and isokinetic knee extensor strength in healthy individuals; however, the correlation coe cient between PA and isokinetic muscle strength in chronic obstructive pulmonary disease patients was reported to be 0.66 [3], which is similar to the results of our study. In a report of muscle EI and MT in isometric strength in young adults, no signi cant correlation was found for muscle EI, while the correlation coe cient for MT was 0.63 [26]. Moreover, the correlation coe cients for isometric strength in the elderly showed no signi cant correlation for EI and 0.48 for MT [26]. In another report, muscle EI was − 0.33 and MT was 0.41 in isometric strength in the elderly. The results of the present study and those of the previous studies showed variability. This could be due to the differences in the measurement positions, target muscles, and muscle contraction types. With regard to the characteristics of the parameters, MT is used as a quantitative method of assessing muscle [17,20]; however, it has been shown that MT also includes connective and adipose tissues [27]. Moreover, the loss of muscle mass alone can only partially explain the muscle weakness [28]. In contrast, PA indicates the structural integrity of the cells [8,9], and muscle EI can distinguish connective tissue and adipose tissue [10], allowing for a qualitative assessment of muscle. However, it has been reported that PA and muscle EI are affected by the water content of the body, speci cally the extracellular water molecules relative to the muscle volume because of their measurement principles [29,30]. Therefore, the parameters PA, muscle EI, and MT in muscle strength evaluation were found to be related, and while the qualitative evaluation method of muscle may be more useful, the correlation coe cients may differ depending on the measurement method and conditions. Therefore, careful interpretation of the data is necessary.
In the present study, PA and muscle EI were signi cantly correlated with maximal gait speed and ve chair stands time in males and females, and with stand-up score in females. In a previous study, the change in PA due to resistance training was reported to have a signi cant positive correlation with the change in muscle quality (muscle strength per unit skeletal muscle mass) [31]. In addition, a study comparing PA in underweight anorexia nervosa (AN) patients, thin individuals, and ballet dancers reported that patients with AN had lower PA than other participants [32], suggesting that there are fewer normal cells with cell membranes of high structural integrity. Another study compared the changes in MT, muscle EI, and motor function in patients with hip osteoarthritis who underwent strength training at high and low velocities [33]. The results showed an improvement in one of the motor function and greater muscle EI of the gluteus maximus in the high velocity group, and no signi cant difference in the changes of other outcome measures between the two groups. In contrast, MT showed no association with any of the motor functions. MT does not represent the cross-sectional area of the entire muscle, but only a portion of it. A previous study that examined the relationship between the change in MT and electromyographic activity in the oblique and transversus abdominis muscles, Hodges et al. [34] described a non-linear relationship, while another study [35] reported that there was no relationship. Considering the above, it can be inferred that PA and muscle EI, which allow qualitative assessment of muscle structural integrity and the amount of non-contractile tissue, could re ect fewer changes in motor function, and were more strongly related to motor function than MT. In terms of usual gait speed, previous studies on PA and muscle EI and usual gait speed found no correlation. It should be noted that the subjects in these studies were elderly [16,36] whereas the healthy young adults in the present study had larger absolute muscle mass. In the stand-up score, 10 out of the 12 males had the highest score, indicating a ceiling effect. Extension of the trunk, hip, knee, and ankle joints occurs during the extension phase of the standing movement [37]. In addition, it is thought that standing up from a low oor requires not only knee extension muscle strength but also combined extension muscle strength of the trunk and lower limbs, thus, it is inferred that young adult males, who generally have more body muscle mass, have a higher complementary function of antigravity movement besides the knee extension movement. On the other hand, elderly people and young adult females have less muscle mass and less complementary function of antigravity movement other than knee extension movement. Therefore, PA and muscle EI in this study were correlated with the stand-up score in females, but not in males.
Our study has some limitations. First, it is unclear whether it is possible to accurately measure PA, which has been found to be highly relevant in postoperative knee patients because they often have metal around the knee joint. Second, it is unclear to what extent the values of parameters such as PA are affected by differences in limb position and muscle contraction style during measurement. Third, since this was a cross-sectional study, causality could not be established, and it is necessary to examine the effects of parameters on muscle strength and motor function longitudinally in the future.

Conclusions
In conclusion, we found an association between isokinetic knee extensor strength, and its alternative assessment methods, and motor function in healthy young adults. PA, muscle EI, and MT, in that order, were signi cantly associated with isokinetic knee extensor strength, and only PA and muscle EI were associated with motor function. The study concludes that a qualitative muscle assessment method could provide a simpler and more multifaceted assessment of muscle strength in healthy young adults.