The relationship between IL-6 and CRP with Sarcopenia in indigenous elderly population at Pedawa Village , Buleleng , Bali , Indonesia

Background: Sarcopenia is a syndrome characterized by decreased muscle mass with decreased muscle strength and or muscle function. Oxidative stress and inflammatory processes are known as triggering factors for sarcopenia by releasing catabolic stimuli of interleukin-6 (IL-6) and C-reactive protein (CRP).This study aims to determine the relationship between IL-6 and CRP levels to sarcopenia parameter such as muscle mass, grip strength, and walking speed. Methods: This study was an analytic cross-sectional design conducted at Pedawa Village, Buleleng District, Bali in August 2016. About 79 respondents aged ≥ 60 years using stratified random sampling technique. The assessed variables were sarcopenia parameter (muscle mass, grip strength, and walking speed) including BMI, as well as IL-6 and CRP levels examination. Spearman and partial correlation test were used to assess the correlation among IL-6, CRP, and sarcopenia parameters. Results: IL-6 levels and CRP were not significantly correlated with the three parameters of sarcopenia. CRP levels correlated with IL-6 (r = 0.37; p = 0.001) and BMI (r = 0.29; p = 0.009). In the male group, IL-6 was only correlated with CRP (r = 0.40; p = 0.011). While in the women group, IL-6 correlated with CRP (r = 0.38; p = 0.017), walking speed (r = 0.33; p = 0.037) and CRP correlated with BMI (r = 0.32; p = 0.049) and total muscle mass (r = -0.32; p = 0.043). After adjustment to BMI variable, IL-6 was correlated with CRP (r = 0,43; p = 0,001) and total muscle mass (r = -0.25; p = 0.026) and significantly correlated in underweight groups (BMI<18.5 kg/m2) (r=-0.50; p=0.026). CRP was not significantly correlated with the three parameters of sarcopenia on Spearman, partial correlation, and Spearman’s specific correlation test based on BMI group. Conclusion: IL-6 levels were associated with total muscle mass loss after BMI adjustmentin Pedawa village’s elderly as a whole. (Health Science Journal of Indonesia 2018;9(1):37-44)

In 2008 to 2040 there was an increase in the world population aged over 65 years by two-fold, from 7.8% to 14.7%. 1 According to the population census in 1990, the number of elderly individuals in Bali reached more than 230,000 people or 8.3 percent of the total Bali population.Then, in 2015 the number of population was expected to increase nearly twice compared to 1990 become more than 432,000 people. 2 In Bali, there are several places with indigenous peoples called the Bali Age Village located in Panglipuran Village (Bangli Regency), Trunyan Village (Karangasem Regency) and Pedewa Village (Buleleng Regency).One of them is Pedawa Village on the top of mountain in North of Bali.Pedawa Village is an isolated and inhabited village of Balinese indigenous people.This village has a specific population where genetic or host factors play an important role in the pathophysiology of a non-communicable disease.Previous research in Pedawa Village regarding noncommunicable diseases related to the aging process obtained the prevalence of underweight by 18.6%, Diabetes Mellitus by 3.9%, systolic hypertension by 14.6% and diastolic hypertension by 12.2%. 3high prevalence of underweight could be high prevalence of sarcopenia.In addition, there is a need for research on non-communicable diseases related to other aging processes, such as sarcopenia to get an overview of the specific elderly population.
The great change that occurs in old age is a progressive decline in skeletal muscle mass, which then leads to a decrease in muscle strength and functional ability.Loss of muscle mass and strength in aging (increasing age) is called sarcopenia.Sarcopenia is a syndrome characterized by a progressive and comprehensive loss of skeletal muscle mass and strength which poses a risk of physical disability, reduced quality of life, and death.There are several mechanisms involved in the onset and progression of sarcopenia. 4 The multifactorial processes that trigger sarcopenia include loss of alpha motor nerve inputs in the cytokines, physical activity, hormonal changes, energy, nutrition intake, oxidative stress, and inflammatory processes.There is evidence that such inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin (IL-6) and C-reactive proteins interact with the hormonal changes inherent to aging, thus causing a decline in physical activity and fat replacement of muscles, which simultaneously lead to volumetric muscle loss. 5,6Subclinical inflammation and oxidative stress in the sarcopenia mechanism trigger the release of catabolic stimuli such as interleukin-6 (IL-6), ciliary neurotropic factor (CNTF) and tumor necrosis factor (TNF).Cellular IL-6 is a significant predictor of sarcopenia. 7IL-6 might trigger muscle atrophy characterized by loss of myofibrillar protein. 6The results of studies in elderly women showed elevated levels of IL-6 due to sarcopenia. 8In addition, there is a significant relationship between high CRP levels and sarcopenia. 9Increased levels of IL-6 and CRP may increase the risk of losing muscle strength. 10sed on the information above, this study aims to know the relationship between IL-6 and CRP levels with the three parameters of sarcopenia such as muscle mass, grip strength, and walking speed in specific population, indigenous and isolated place.This study is still rarely performed or published in Indonesia.The results of this study are expected to be a reference for preventive and curative interventions of sarcopenia in the future.

Study design and samples
This cross-sectional analytic study was conducted in Pedawa Village, Buleleng District, Bali in August 2016 using stratified random sampling of 600 elderly population spread over 6 hamlets.The respondents were people with greater age or equal 60 years of cooperative attitudes without any comorbidities enrolled in the study (n = 79).The exclusion criteria include subjects with malignancy; suffering from acute infection; heart failure; chronic liver disease; kidney failure stage 4 and 5; taking corticosteroids; aspirin or NSAIDs for at least two weeks; immobility or Barthel Activity Daily life scores less than 9; suffering from sequelae; severe cognitive damage; and experience an inability to be interviewed due to socio-linguistic problems and / or aphasia.Other comorbidities were controlled by analysis.The elderly individuals were explained the purpose of the study and had obtained informed consent.

Data collection
Data were collected through interview method, anthropometric and Bioelectrical Impedance Analysis (BIA) measurement, grip strength, walking speed, examination of IL-6 and CRP serum levels at one time.

Anthropometry and body fat percentage
The anthropometric examination was performed by body weight (BW) and height (H) measurement based on knee height.Body height measurement (cm) in the elderly using knee height was carried out by knee height gauge, then converted through calculation of Chumlea knee height formula. 11The waist and hip circumference were measured using a cloth tape measure in centimeters (cm).BMI was calculated using the Omron BIA then categorized based on WHO criteria with some modifications into three groups namely underweight (BMI < 18.5 kg/m2), normal (18.5 < BMI < 24.9 kg/m2), as well as overweight and obese (BMI > 25 kg/m2). 12The body fat percentage was obtained through BIA measurement.

Sarcopenia parameter
Based on the Asian Working Group for Sarcopenia (AWGS) recommendation, sarcopenia parameters measured in this study were muscle mass, grip strength, and walking speed. 11The muscle mass measurements using BIA tools obtained total skeletal muscle mass (upper limb muscle mass, lower limbs, and trunk) in percent (%).The grip strength (muscle power) examination was performed with the aid of a handheld dynamometer, expressed in kilograms (kg).Walking speed (m / s) was calculated with a run test as far as 4.57 meters.

IL-6 and CRP levels
The levels of IL-6 and CRP were derived and examined from blood serum.Approximately 5 mL of venous blood subject taken by researchers assistant (phlebotomist).The procedure was performed by a qualified professional in the morning (between 8:00 and 10:00) to avoid the influence of circadian cycle changes.The blood in the serum centrifuge is kept below 0°C by dry ice and taken to the laboratory.IL-6 and CRP concentrations were analyzed using the Quantikine HS Human IL-6 Immunoassay reagent (HS Catalog of HS600B) and Quantikine Human CRP Immonoassay (Catalog of DCRP00) Examination of IL-6 and CRP using Quantikine HS Human IL-6 Immunoassay (HS600B Catalog Listing) and Quantikine Human CRP Immonoassay (DCRP00 Catalog Number) were measured by Enzyme-linked immunosorbent assay (ELISA) method with pg / mL unit.This measurement is done one time without repetition.

Statistical analysis
The descriptive statistical analysis was performed using measures of central tendency (mean and median) and variability (range and standard deviation).The data distribution wasn't normal so that Spearman correlation test was used to measure the correlation between numerical variables.A partial correlation test was applied to determine the correlation between IL-6 and CRP to the sarcopenia parameters by adjusting the BMI variable, considering a significance level (α) of 0.05.Statistical analyses were carried out using the Statistical Package for Social Sciences.(SPSS; version 16.0, Windows environment).28.68 ± 7.82 Visceral fat (%) 2 7.90 ± 6.25 Total skeletal muscle mass (%) 2 89.03 ± 1.46 Upper Limb (%) 2 30.40 ± 5.96 Lower Limb (%) 2 39.33 ± 6.14 Truncal (%) 2 19.30 ± 3.85 Grip strength (kg)

The correlation between IL-6 and CRP with component and related sarcopenia parameters
The study found no significant correlation between IL-6 and CRP with sarcopenia parameters (muscle mass, grip strength, and walking speed) in Spearman correlation test, but obtained a significant correlation between IL-6 with CRP (r = 0.37, p = 0.001) and CRP with BMI (r = 0.29, p = 0.009).(Table 2 and 3) In each sex group, a correlation analysis of the sarcopenia parameter was performed.In the male sex group, there was a significant correlation between IL-6 level and CRP (r = 0.40, p = 0.011).whereas in the female sex group there was a significant correlation between IL-6 and CRP levels (r = 0.38, p = 0.017), IL-6 with walking speed (r = 0.33, p = 0.037), CRP with BMI (r = 0.32, p = 0.049), and CRP with total skeletal muscle mass (r = -0.32,p = 0.043).(Tables 4 and 5) The scatter plot diagram aims to determine the relationship pattern between BMI and IL-6 as well as to predict whether a non-significant relationship between IL-6 and the three sarcopenia parameters are due to the presence of the BMI variable.The diagram showed that high distribution of IL-6 appears trend at low and high BMI values (Figure 1).

Correlation between IL-6 and CRP on specific sarcopenia parameters based on BMI group
Based on the assumption that BMI variable influences a significant correlation between IL-6 and CRP with sarcopenia parameter, partial correlation test was performed between IL-6 and CRP with related sarcopenia parameter by adjusting BMI variable.Partial correlation test by adjusting BMI suggest that IL-6 variable still showed significant correlation with CRP (r = 0.43, p <0.001) (Table 6).The result of partial correlation test between IL-6 and related sarcopenia parameter after adjusting for BMI variable showed significant correlation to total skeletal muscle mass.but CRP is not significantly associated with sarcopenia parameters.Then, we performed a separate Spearman correlation test based on BMI group (underweight, normal, and overweight-obese) (Table 7).In all three BMI groups, there is a significant negative correlation between IL-6 and total skeletal muscle mass in the underweight group (P< 0.05).

DISCUSSION
Sarcopenia associated with aging is a slow, progressive, and seemingly inevitable process.This directly affects the functional capacity of elderly individuals and leads to a decline in health.This condition will directly affect the functional capacity of elderly individuals and lead to a decrease in health. 13ble 7. Correlation between IL-6 and CRP with specific sarcopenia parameters in BMI group This study aims to determine the relationship between IL-6 and CRP with sarcopenia parameters.Prior to statistical adjustment for BMI variables, we found no significant correlation between IL-6 and CRP with three sarcopenia parameters.To see more detailed results we do analysis by sex.In the male sex group, there was a significant coefficient on IL-6 with CRP.In the female sex group, there was a positive correlation between IL-6 with CRP, walking speed and CRP with BMI and negative correlation with CRP with skeletal muscle mass.A study conducted on 130 elderly women found a significant correlation between IL-6 levels with functional muscle. 14In the study by Choi et al (2013), it was found out that BMI was associated with CRP levels and the relationship was greater in women than in adult males. 15The negative association between CRP levels and skeletal muscle mass was found in a study of 118 elderly women and was found to be significantly higher in CRP in older people with lower muscle mass than those with normal muscle mass. 16CRP affects muscle cell size through the MPS pathway mechanism. 16ter adjusting the BMI variable, partial correlation test was obtained a significantcorrelation between IL-6 and muscle mass (weak negative correlation).These results are consistent with research conducted by Haren et al (2010) who found that an increase in IL 6 leads to a loss of muscle mass. 17However, the study also showed that elevated levels of IL-6 will lead to decreased muscle function, while our study found no association between IL-6 and muscle function (walking speed). 17e of the underlying mechanisms of mass muscle reduction in high-IL-6 levels is muscle atrophy induced by elevated levels of IL-6. 18Besides the effect of muscular atrophy, another theory that allegedly underlies decrease of mass muscle decline is a catabolic state induced by IL-6 in muscle.However, this catabolic state is often found only in sophisticated systemic conditions such as sepsis or cachexia. 18,19 our specific analysis based on BMI groups, a significant relationship between IL-6 and muscle mass was consistently found in the BMI underweight group (moderately negative correlation).Nonsignificant relationships were found in normal BMI and overweight-obesity groups.The high IL-6 distribution in subjects with low BMI scores was depicted in the scatter plot chart.This is an important note to us that keeping the elderly from underweight conditions will allow them to avoid the muscle mass loss due to IL-6, so as to prevent the onset of sarcopenia.These data can be a reference for clinicians to consider the BMI modification in elderly individuals.
IL-6 was significantly associated with CRP (positive correlation) in partial correlation test by adjusting BMI and Spearman correlation test without adjusting for BMI variables.This result is similar to a study performed by Il'Yasova, et al. (2008), which suggests a positive correlation between IL-6 and CRP levels. 20L-6 is responsible for triggering the synthesis of acute phase proteins such as CRP theoretically. 21owever, the correlation between IL-6 and CRP did not become significant when a specific analysis was performed based on the BMI group in our study.
Although there is a correlation between IL-6 with a reduction in CRP, our study did not find any correlation between CRP and the sarcopenia parameters, particularly in muscle mass and grip strength.This indicates that CRP does not mediate the role of IL-6 in the mass muscle decline.The results of our study differed from a prior study conducted by Atkins et al. (2014),who reported that CRP levels were positively related to low muscle mass, regardless of the effect of age, lifestyle, and body composition. 22In addition, it is also demonstrated that there was a pivotal role of increased CRP level in the muscle strength reduction. 9e conflicting results observed in studies are probably due to the differences in sample characteristics, such as comorbidities, socioeconomic status, education level, and physical activity level.In this study, the sample size is relatively small (n = 79) that a specific correlation test based on BMI group could not be carried out optimally in each group of BMI.This can be seen from the inconsistent relationship between IL-6 and CRP in which the correlation between the two variables becomes insignificant when a specific correlation test was performed for each BMI group.Therefore, similar studies in the future are expected to use larger samples.Another our study limitation is the exclusion of several variables suspected to be associated with sarcopenia parameters into data analyzes such as nutritional intake (protein, fat, carbohydrates) and the amount of energy/calorie use.In this context, we did not explicitly perform a linear regression to determine the relationship between nutritional intake, energy use, IL-6, and CRP with sarcopenia parameters.Besides, the cross-sectional design does not allow causal inferences about the relationships between the studied variables.
In conclusion, in the elderly men group IL-6 correlated with CRP, whereas in the elderly women group IL-6 had a positive correlation with CRP, BMI and walking speed and CRP negatively correlated with total muscle mass.IL-6 levels were correlated with the decrease in total muscle mass of Pedawa village elders as a whole after adjustment of BMI variables.

Table 1 .
). Sample Characteristics of Study

Table 2 .
Correlation between IL-6 levels with component and related sarcopenia parameters.

Table 3 .
Correlation between CRP levels with component and related sarcopenia parameters.

Table 4 .
Correlations between IL-6 levels and CRP with sarcopenia parameters in elderly men

Table 5 .
Correlation between IL-6 levels and CRP with sarcopenia parameters in elderly women