Hyperhomocysteinemia Associated with Low Muscle Mass, Muscle Function in Elderly Hemodialysis Patients: An Analysis of Multiple Dialysis Centers

Background The hyperhomocysteinemia was with high prevalence and has been considered as a risk factor for cardiovascular disease in hemodialysis patients. These patients also experienced a high risk of muscle wasting caused by the comorbidity, malnutrition, and low physical activity. We investigated the associations of homocysteinemia with muscle mass, muscle function in elderly hemodialysis patients. Methods A clinical cross-sectional study was conducted on 138 hemodialysis patients aged 65 years and above in seven hospital-based hemodialysis centers in Taiwan. The data on anthropometry, laboratory, and 3-day dietary intake was examined. The skeletal muscle mass (SMM) was measured by the bioelectrical impedance analysis; the SMM was adjusted by height or weight as SMMHt2 (kg/m2) and SMMWt (%). Muscle function was defined as handgrip strength (HGS) (kg) measured by handgrip dynamometer. Statistical analyses were conducted using simple regression and multivariable stepwise regression analysis. Results In the total sample, 74.6 % of hemodialysis patients were hyperhomocysteinemia (≥ 15 μmol/L). The means of SMMHt2, SMMWt, arm lean mass, hand grip strength, and muscle quality were 8.7 ± 1.2, 37.7 ± 5.6, 1.7 ± 0.5, 21.1 ± 7.4, and 10.0 ± 3.0, respectively. The multivariable stepwise regression analysis showed that homocysteinemia level was significantly inversely associated with SMMWt (B-coeff. = -0.03, p = 0.02) in hemodialysis patients above 65 years old, but not with muscle function. Conclusions Hyperhomocysteinemia is common and associated with decreased muscle mass in the elderly hemodialysis patients. Future studies are suggested to explore the impact of the homocysteine-lowering therapy on muscle decline.


Introduction
The United States Renal Data System reported that the prevalence of treated end-stage renal disease (ESRD) was highest for individuals aged 65-74 years in Taiwan [1]. The elderly hemodialysis patients experienced the high risk for muscle wasting, which was related to higher morbidity and mortality [2]. Muscle wasting was the most common condition in elderly people, especially those undergoing hemodialysis with a high prevalence of decreased muscle mass and muscle function, varying from 12.7% to 45.1% in hemodialysis patients [3], the declines in muscle mass and function caused by age [3], comorbidity like diabetes mellitus and infections [4], malnutrition [5], and physical inactivity [6]. In addition, physicians prescribed exercise restrictions in those patients with complications during the treatment [7]. Furthermore, declines of muscle mass and function were exaggerated due to the long bedridden time for the dialysis, low physical function [8], low exercise capacity [9], and increased muscle atrophy [6].
The hyperhomocysteinemia was presented in 80% to 90% of hemodialysis cases [10]. Homocysteine (Hcy) has been considered an important cardiovascular risk factor [11]. Moreover, studies have established an association between high levels of homocysteine and a decline in physical function in elderly populations [12]. The lower muscle mass was observed in higher plasma homocysteine group in ≥ 65 years adults [13]. The effects of hyperhomocysteinemia in vascular and myocyte function leading to impaired muscle function were summarized as follows: (i) oxidative defense reduced and production of reactive oxygen species enhanced, (ii) inhibition of nitric oxide (NO) signaling, (iii) inflammation and its associated changes, and (vi) endoplasmic reticulum (ER) stress enhanced [14]. Previous empirical studies have shown elevated Hcy associated with the decline of muscle strength and physical function in the older adults [15,16].
Since hemodialysis patients are with a high prevalence of hyperhomocysteinemia and in a high-risk group of muscle wasting, however, there are a limited number of studies that discussed this issue in hemodialysis patients, especially in the elderly. Therefore, we aim to investigate the association between homocysteine level and muscle mass, muscle function in elderly hemodialysis patients. All patients signed written informed consent forms before their participation.

Materials and Methods
The total sample of 138 patients aged 65 years and above and undergoing hemodialysis was recruited for the study. Patients who received stable hemodialysis treatment in the previous 3 months with equilibrated Kt/V of 1.2 and higher were included. Patients with obvious edema, hyperthyroidism, hypothyroidism, amputation, malign tumor, pregnancy, or hospitalization for renal disease reason were excluded.
. . Data Collection . . . Demographics Data. We conducted chart reviews to collect the data related to age, gender, dialysis vintage, diabetes mellitus (DM), hypertension, cardiovascular disease (CVD), and the anthropometry data including dry weight, height, and interdialytic weight gain.
. . . Physical Activity. Patients' physical activity was assessed using the short version of the International Physical Activity Questionnaire (IPAQ) [17]. Interviewers recorded the average number of days per week and the average time per day that patient spent on exercising (vigorous, moderate, or walking exercise) in the past 7 days. The value of metabolic equivalent (MET in kcal/day) value was used to examine the levels of physical activity [17].
. . . Dietary Intake Data. All patients wrote down a threeday dietary record, including one dialysis day, one nondialysis day, and one day during the weekend. The data collection of dietary intake was also mentioned in our previous publication [18]. In brief, qualified dietitians taught patients how to fill in the record. To assure the record, the well-trained dietitians contacted with all patients and conducted the interviews by face-to-face, or by telephone. Next, the dietitians used the 24 h recall to confirm the data provided by patients using the common utensils in the household as the means. The nutrients were then analyzed using nutrients analysis software (e-Kitchen, Taichung, Taiwan) based on Taiwanese nutrition compositions as the nutrient database.
. . . Biochemical Values. We collected the 8-hour fasting and predialysis blood samples and sent to the Laboratory Department in Taipei Medical University Hospital for analyzing the biochemical parameters. The following parameters were collected by reviewing patient medical charts: total cholesterol, creatinine, and fasting blood glucose.
. . Measurements . . . Homocysteine Measurements. Homocysteine was measured used in the enzymatic method. 8-hour fasting and predialysis blood samples were withdrawn and collected in EDTA blood collection tubes keep tube cold at 4 ∘ C and centrifuged within 1-2 hour, then plasma was assessed using the Roche Cobas c702 automatic analyzer (Rui An international Co. Ltd., Taipei, Taiwan) at the of the Laboratory Department in Taipei Medical University Hospital.
. . . Other Biochemical Measurements. The 8-hour fasting and predialysis blood samples were analyzed using standardized protocols for serum albumin, insulin, serum folate, serum vitamin B 12 , high-sensitivity C-reactive protein (hs-CRP), thyroid-stimulating hormone, and plasma homocysteine. The cutoff values for folate and B 12 were 3 ng/mL and 250 pg/mL, respectively. Additionally, insulin resistance was measured by the homeostasis model assessmentestimated insulin resistance (HOMA-IR) calculated as (glucose [mg/dL] × insulin [ U/mL)]/405 [19]. In the clinical assessment of nutritional status, we calculated the geriatric nutritional risk index (GNRI) as described previously, when GNRI < 91.2 exhibited a poorer nutritional status. Hypoalbuminemia and inflammation were defined as serum albumin less than 3.5 g/dL and hs-CRP higher than 0.5 mg/dL, respectively. Normalized protein nitrogen appearance (nPNA) ≥ 1.2 g/kg was appropriate protein intake in hemodialysis patients as suggested by the National Kidney Foundation [20].
. . . Anthropometry Measurements. Skeletal muscle mass (SMM) and body fat are measured by using the bioelectrical impedance analysis (InBody S10, Biospace, Seoul, Korea) after the hemodialysis session (sitting position). The eight surface electrodes are placed on the thumbs, middle fingers, and either side of the ankles of the patients using multiple operating frequencies of 1, 5, 50, 250, 500, and 1,000 kHz. Moreover, SMM was normalized for weight as SMM Wt (%) and height as SMM Ht2 (kg/m 2 ), which are indicators of muscle mass. Muscle quality (MQ) is an important determinant of muscle function, defined as muscle strength or power per unit of muscle mass and calculated as the ratio of hand grip strength (kg) to arm lean mass (kg) [21]. The handgrip strength (HGS) was measured before a hemodialysis session using grip strength dynamometer (Jamar, Sammons Preston, Bolingbrook, IL) with a precision of 0.5 kg. During the measurement, patients were asked to stand straight with arm and hands being neutrally hung beside the body and then use maximum effort to squeeze the dynamometer with nonfistula hand for at least 3 seconds, and each time was provided at least 10 seconds for recovery. Patient performed the handgrip for 3 times and the pick performance was noted as the final result.
. . Statistical Analyses. The sample of 138 patients with data of muscle mass and a sample of 87 patients with the data of muscle function were analyzed. The descriptive analyses were used to illustrate the mean ± standard deviation, percentage of social demographics, biochemical parameters, dietary intake, and patients' characteristics. Simple regression was used to identify the predictors of muscle mass and muscle function which were known as prognostic factors, statistical significance when p < 0.05. Multivariate linear regression models were performed to investigate the associations of Hcy and the muscle mass and muscle function. The variables showed the associations with muscle mass and function at p < 0.2 in the simple regression analyses which were kept in the multivariate models. Variables such as age, gender, and energy intake were also included in the analysis. The unstandardized regression coefficient (B), 95% confidence interval, and adjusted R square values were presented appropriately. Statistical analyses were performed using SAS software (ver. 9.4; SAS Institute Inc., Cary, NC, USA).

. . Associated Factors of Muscle Mass and Muscle Function.
The SMM, SMM Ht2 , and SMM Wt were positively correlated with creatinine and height and were negatively correlated with age, gender, and body fat. HGS was positively associated with creatinine and height and was negatively correlated with gender. Muscle quality (MQ) was negatively correlated with body weight and BMI ( Table 2).
The results stepwise regressions show that SMM was significant associations with energy intake (regression coefficient, B = < 0.  (Table 4).

Discussion
The current study found that Hcy was significantly inversely associated with muscle mass in hemodialysis patients aged 65 years and above. Besides, higher Hcy was marginally associated with muscle function decline in hyperhomocysteinemia hemodialysis patients aged 65 years and above. A previous longitudinal study has confirmed the association between higher homocysteine lower muscle strength in a general population aged 50 years or older [22]. Contributing factors such as age, low physical performance, and the presence of comorbidities were associated with muscle wasting [23].
The previous studies showed that elevated Hcy is associated with muscle function declined in the older adult [15,16]. In addition to multiple hypotheses hyperhomocysteinemia impaired muscle function concluded by Veeranki and Tyagi [14], Hcy thiolactone reacts with proteins by a mechanism involving homocysteinylation of protein lysine residues in human serum and leads to protein damage and atrophy of skeletal muscle [24], the phenomenon called protein homocysteinylation [24]. Protein homocysteinylation is the posttranslational acylation of free amino groups (e.g., protein-Nhomocysteinylation) or formation of a covalent-S-S-bond (protein-S-homocysteinylation) mediated by Hcy thiolactone [24], which potentially causes significant alterations in the protein function. A study reported that protein-Nhomocysteinylation and protein-S-homocysteinylation were significantly higher in hemodialysis patients, and the significant association between plasma Hcy and protein-Shomocysteinylation was also found [25]. This indicated that hyperhomocysteinemia may cause the toxicity via the oxidative damage to proteins [26] and may result in muscle weakness and atrophy [24].
In our study, Kt/V was significantly associated with the lower SMM in hemodialysis patients. The negative association between Kt/V and muscle mass was also demonstrated in 34 Japanese hemodialysis patients [27]. The result indicated that patients with lower muscle mass may require a higher dialysis clearance. Therefore, muscle mass should be addressed while evaluating the hemodialysis adequacy.
The present study showed that dialysis vintage, creatinine, and BMI were independently associated with muscle mass and/or muscle function. The predialysis serum creatinine level will be proportional to dietary protein intake and the SMM that lower muscle mass may indicate the lower creatinine excretion [28]. The previous study showed that   the hemodialysis vintage was negatively associated with the loss of SMM and muscle function. In addition, BMI was positively associated with SMM [27]. The possible reason for the negative correlation between BMI and MQ was supposed to be the calculation formula, BMI would be increase, and MQ would be decreased when skeletal muscle mass increases. Our study shows that body fat was an independent determinant of muscle mass and muscle function among hemodialysis patients. A previous study was conducted using the computed tomography (CT) to take mid-thigh muscle cross-sectional area in a 36-year-old hemodialysis patient and an 80-year-old hemodialysis patient. Results showed that the fat accumulated in the striated muscle cells and intermuscular adipose tissue in older hemodialysis patients. Besides, the results also reveal that reduced muscle cross-sectional area, increased intermuscular adipose tissue, and/or intramuscular lipid were independently associated with aging and indices of maximal strength and physical performance [29].
Our study was with a number of strengths and limitations. The plasma homocysteine was analyzed by the international standard protocol in the hospital. We used the bioelectrical impedance analysis (BIA) as body composition measurement. The BIA is inexpensive, portable, and easyto-use as compared with others devices. The measurement conducted by BIA might be minor differences as compared with other methods such as the dual-energy X-ray absorptiometry (DEXA), CT, and magnetic resonance imaging (MRI). However, the examination of total-body muscle mass using the BIA was highly correlated with estimation using the MRI in hemodialysis patients [30]. Finally, the basis of a cross-sectional design cannot provide the causal evidence between plasma Hcy and muscle mass. Therefore, the results must be interpreted with caution. Future cohort designs or control trials are suggested.

Conclusion
The current study revealed that hyperhomocysteinemia is high prevalence in hemodialysis patients. Elevated homocysteine level is associated with lower muscle mass in hemodialysis patients aged 65 years and above. Thus, the homocysteinelowering therapy might have a positive impact on muscle mass. The longitudinal studies are suggested to provide a deeper understanding of the association. Randomized control trials are needed for evaluating the effectiveness of therapy.

Data Availability
Data is available upon request to corresponding author of this article.

Disclosure
The funder had no role in the decision to collect data, data analysis, or reporting of the results.