Background

The bioelectric impedance analysis is widely used for body composition and nutritional status assessment due to its simple, rapid, non-expensive, reproducible and non-invasive method. Beyond body composition, another important parameter obtained by bioelectrical impedance analysis (BIA) is the phase angle (PhA) value. PhA is the arc-tangent value from reactance and resistance and is considered an indicator of body cell mass and cell membrane integrity [1].

PhA has been used as a supporting tool for nutritional diagnostic in clinical practice and is used as a predictor of membrane integrity and cell mass [2]. Additionally, PhA has been associated with a number of clinical conditions, such as advanced liver fibrosis in patients chronically infected with hepatitis C virus [3], cancer [4], peritoneal dialysis [4], malnutrition [5], low muscle mass [6, 7], and higher mortality in patients with several diseases [8,9,10]. Recently, in hemodialysis patients, it was also observed that PhA values were associated with muscle function and overall quality of life [11].

Metabolic syndrome (MetS) is a clinical condition that predisposes the development of cardiovascular diseases and type-2 diabetes and is associated with higher risk of all-cause mortality [12]. MetS is closely related to increased body fat mass, visceral fat deposition, and insulin resistance, which constitute clinical conditions that require nutritional care. Additionally, MetS pathophysiology includes several comorbities such as inflammation, oxidative stress, pro-thrombotic, and pro-atherogenic processes [13,14,15,16].

As PhA value is a useful tool for identifying cell membrane integrity dysfunction and it is known that MetS increases oxidative stress and inflammation, which consequently can lead to cellular damage [15], we hypothesized that MetS and some blood markers related with this clinical condition could be associated with lower PhA values. Therefore, the aim of this study was to identify the association of PhA with MetS and blood markers in individuals clinically screened for a lifestyle modification program.

Methods

Individuals

A cross-sectional study was conducted with individuals enrolled in the lifestyle-modification program “Mexa-se Pró-Saúde (Move for Health)” in Botucatu City, Brazil. This program offers primary care for non-communicable chronic diseases by promoting nutritional counseling and regular physical activity. Inclusion criteria were 35 years or older adults without metabolic or motor disabilities that could limit the practice of exercises. Exclusion criteria were the presence of other inflammatory diseases, such as cancer and rheumatoid arthritis; use of anti-inflammatory drugs; and those that did not realize all assessments. From a total of 587 subjects, 170 did not perform all assessments or were excluded during clinical evaluation. Then, 417 subjects (76 men and 341 women, 53.9 ± 9.4 years old) were studied. All subjects had signed an informed consent and the study was approved by the Human Research Ethics Committee of Botucatu Medical School (FMB), São Paulo State University (UNESP, Brazil) in accordance to the “Declaration of Helsinki” (Research Ethics Committee protocol 4049-2011).

Assessments

Body mass (kg) was measured by anthropometric scale platform (Filizola®) and height (m) was determined using a portable stadiometer (Seca®). Body mass index (BMI) was calculated (weight (kg)/height (m)2) and classified according to the World Health Organization [17]. Waist circumference (WC) was measured using the Sanny® steel anthropometric tape positioned at the midway point between the last rib and the iliac crest [17].

Whole-body bioelectrical impedance analysis was performed using a tetrapolar bioimpedance (Biodynamics®, Model 450, USA) applying an alternating electric current of 800 μA and 50 kHz. The measurements were in accordance with the procedures described by the National Institutes of Health Technology Assessment Conference Statement [18]. PhA in degrees (o) was calculated by using the equation (reactance/resistance) × (180/л). Since there is no consensus for PhA cutoff, we decided to use the PhA median value of our sample (6.3°) as the cut point, as described before [6]. The same cutoff was used for men and women since we tested and found no difference in PhA values according to sex (men 6.8 ± 0.8° vs. women 6.3 ± 0.8°, p = 0.57). This median value is supported by another study evaluating the same age group [19].

Subjects were previously instructed to not perform vigorous physical exercises for 24 h, to avoid alcohol and caffeinated drinks for 72 h before testing and previous emptying of the bladder. Women that were not at post-menopausal period were instructed to avoid menstrual period for analysis. Individuals with abnormal hydration status were excluded considering the normal values of total body water per lean mass of 69 to 75%, according to the bioimpedances’s manufacturer recommendations, ensuring greater reliability in the PhA values. Muscle mass was estimated using Janssen et al.’s equation [20] and muscle mass index (MMI) was calculated as MM (kg)/height2 [21].

Subjects were submitted to clinical evaluation for assessments of systolic (SBP) and diastolic (DBP) blood pressures as described previously [22].

Blood samples were collected after an overnight fasting in the same morning that BIA evaluation was performed. Laboratory analyses of lipid parameters (total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, and triglycerides), glucose, uric acid, urea, γ-glutamyl transferase (gamma-GT), and albumin were measured by dry chemistry (Vitros® 5600, Ortho Clinical Diagnostics, Johnson & Johnson Company, Raritan, NJ, USA) within 4 h after blood collection. Serum C-reactive protein (CRP) concentrations were measured by high-sensitivity immuno-nephelometric assay (Siemens Healthcare Diagnostics, Marburg, Germany).

MetS diagnosis was in accordance with NCEP-ATPIII criteria [23]. High waist circumference (WC >102 cm for men and >88 cm for women), hyperglycemia (FBG ≥100 mg/dL), hypertriglyceridemia (TG ≥150 mg/dL), reduced HDL-C (<40 mg/dL for men and <50 mg/dL for women), and hypertension (SBP ≥130 mmHg and/or DBP ≥85 mmHg) were considered as MetS components. MetS was diagnosed when three or more of these components were altered [23]. Low-grade inflammation was determined when CRP values were >3.0 mg/L [24]. Abnormal values of total cholesterol (≥200 mg/dL) and LDL cholesterol (≥130 mg/dL) were set according to the last Brazilian guidelines for dyslipidemia and atherosclerosis prevention (2013) [25] .

Statistical analyses

Statistical analyses were performed at SAS software, version 9.1.3. Descriptive data were presented as mean ± standard deviation (parametric variables) or median and interquartile range (non-parametric variables). We tested the differences between lower PhA (≤6.3°) and higher PhA (>6.3°) groups for parametric variables using independent t test, whereas for non-parametric variables using Mann-Whitney’s test. Chi-square was performed to evaluate the prevalence of MetS according to PhA groups. One-way ANOVA was performed to compare PhA values according to the number of altered MetS components. Logistic regression was used to determine the odds for lower PhA (≤6.3°) according to MetS and blood biomarkers, adjusted for age, sex, BMI, and muscle mass index. Statistical significance was set as 5% (p < 0.05) for all analyses.

Results

Among the entire sample, 28% of individuals were of normal weight; 30% overweight; and 42% obese. The total proportion of individuals with MetS was 52%, whereas in the higher PhA values group, MetS prevalence was 44% and in the lower PhA values, it was 56%, but no differences were found between groups (p > 0.05). Additionally, there was no difference in PhA values according to the increasing number of MetS components (Fig. 1).

Fig. 1
figure 1

PhA mean values according to the number of metabolic syndrome components. p > 0.05

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Subjects with lower PhA values were older and showed lower BMI, WC, MMI, and diastolic blood pressure. The HDL cholesterol and gamma-GT concentrations were significantly higher in those with lower PhA values, whereas creatinine and uric acid concentrations were significantly lower in this group (Table 1). Additional individual’s characterization can be found in our previous research [6].

Table 1 Subject’s characterization according to the lower or the higher phase angle (PhA) values
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Logistic regression analysis showed that higher CRP concentration was the only parameter associated with lower PhA (Table 2). Those individuals with CRP ≥3.0 mg/L showed 62% more chance of having lower PhA values even after adjustment for age, sex, BMI, and MMI (OR = 1.62; 95%CI: 1.01–2.60). The presence of MetS and the other biochemical parameters showed no association with lower PhA values in all models analyzed.

Table 2 Logistic regression analysis between lower phase angle (PhA) values and Metabolic syndrome and blood markers
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Discussion

The main finding of this research was that higher concentrations of CRP increased the odds of low PhA regardless of sex, age, BMI, and MMI. Furthermore, contrary to our hypothesis, MetS was not associated with PhA. To the best of our knowledge, this is the first study showing such associations in individuals clinically screened for a lifestyle modification program.

A lower PhA value is considered a marker for reduced cell membrane integrity [2]. The inflammatory process is a well-known condition related to tissue injury and consequently cell membrane integrity disarrangement [26], and several studies with different clinical conditions also reported associations between PhA values and inflammatory markers [27,28,29]. Interestingly, adipose tissue seems to be the greatest target for leukocyte infiltration and also a source of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) [30]. TNF-α is a well-recognized apoptosis inductor, and more recently, a mechanistic process involving cell membrane integrity induced by activation of the TNF-α receptor was reported [31]; however, in our data, inflammation was associated with PhA independently of BMI. Additionally, we adjusted for BMI, and not for waist circumference, due to our research group has already shown that waist circumference is not associated with PhA [6]. Another hypothesis for the inverse association between PhA and inflammation could be an indirect effect of oxidative stress. High CRP concentrations is related to increased free radicals [32], which can lead to cellular damage [33] and probably could decrease PhA values. Although we have not evaluated specific oxidative stress markers, PhA was not associated with uric acid, that is responsible for 2/3 of total plasma antioxidant capacity [13]. Therefore, the probably mechanism for the observed relation between CRP and PhA may not be dependent of the increased oxidative stress; however, this affirmation cannot be concluded only based on uric acid concentration and more studies are necessary to evaluate the influence of oxidative stress on PhA.

In our descriptive analysis, it was noted that individuals with higher PhA values showed higher levels of uric acid and lower HDL-c. However, these biochemical variables were not associated with PhA, which shows that individuals with higher values of PhA also had higher waist circumference and BMI [6], and these biochemical variables were higher due to higher amount of adiposity in this group [34, 35]; therefore, there were no direct association between PhA with uric acid and HDL-c.

The presence of MetS is well recognized as related to low-grade inflammation and insulin resistance development [14]. However, even though the CRP were associated with PhA, as demonstrated, PhA values were not associated with the increasing number of MetS components and with other blood biomarkers. Recently, a cohort study also did not find association between some MetS components and PhA values [36], reinforcing our findings. Therefore, our data suggests that PhA values could be associated with an unspecific pro-inflammatory process and may be a useful tool for indirectly accessing it, independently of the presence of MetS.

We evaluated the odds to present PhA lower than 6.3°, a value that corroborates with the findings of Barbosa-Silva et al. (2005) [19] who studied the same age group of healthy subjects and found values of 7.3 ± 0.89° (6.12°; 8.68°) for men and 6.5 ± 0.87° (5.48°; 7.96°) for women. Although it is known that PhA values are higher in men [19], the median values were not different between sexes in our population. However, to avoid the influence of sex in our results, all analyses were adjusted for this variable. Other important adjusted variable was age, which is also known to be related with PhA [19] and MetS [37]. Additionally, a recent study [38] showed that age, fat-free mass, and height are the most important variables that explain PhA variances in healthy individuals. It is important to remember that we adjusted the analysis for muscle mass, which was estimated by an equation that has resistance value as one of the variables, such as PhA calculation. Therefore, this does not seem to be a limitation in our data because even after adjustments for MMI, the association remained significant, which showed that MMI did not indirectly adjusted PhA itself. Therefore, we showed that CRP was associated with PhA independently of these important confounding variables.

Some limitations should be illustrated at present study. This is a cross-sectional study; therefore, the cause-effect relationship between CRP and PhA values cannot be determined. Furthermore, our sample had higher proportion of females and is not representative of a larger community. This difference occurred because all subjects were spontaneously engaged with a lifestyle change program, and it is known that women are more concerned to change their lifestyle than men [39]. However, all analyses were adjusted for sex, ensuring the statistical control for this discrepancy. In addition, only one inflammatory marker was evaluated. Although CRP is considered the most powerful unspecific marker for inflammation, we did not analyze TNF-α, a pro-inflammatory cytokine that seems to be more related with cell plasma membrane instability [31]. However, we reinforce that this was the first study reporting the association between PhA values and low-grade inflammation marked by plasma CRP concentrations, independently of body composition and MetS diagnosis. Specificity and sensitivity of PhA values as well as cut points for detect inflammatory processes still need to be determined and should be carried out in future studies.

Conclusions

In conclusion, higher C-reactive protein concentrations increased the odds of low PhA independently of the presence of MetS. Additionally, contrary to our hypothesis, MetS was not associated with PhA values. Longitudinal studies are needed to confirm these results.