**3. Results**

Table 1 shows the participant characteristics. The participants had an average age of 64.24 ± 10.4 years; 128 were male and 178 were female. The mean albumin serum level was 4.39 ± 0.25 (g/dL); The mean f(HMA) was 69.49 ± 7.02%; there were 151 and 155 participants in the N and L groups, respectively. With respect to the severity of LS, 118 participants (38.6%) were at no risk (stage 0), 116 (37.9%) were stage 1, and 72 (23.5%) were stage 2 [43].


**Table 1.** Demographics and clinical characteristics.

BMI: body mass index; f(HMA): fraction of human mercaptalbumin; f(HMA) = HMA area/(HMA area + HNA area); HMA: human mercaptalbumin; N: participants with f(HMA) of 70% or more; L: participants with f(HMA) less than 70%; LS: locomotive syndrome. Values are mean ± SD for each group.

#### *3.1. Non-Elderly Participants*

The average age was 54.19 ± 7.34 years, and the mean f(HMA) was 72.96 ± 5.86%. Eighty-four (67.7%) and 40 (32.3%) subjects were considered to be in the N and L oxidative stress groups, respectively. In terms of LS, 52 (41.9%), 50 (40.3%), and 22 participants (17.8%) were grouped into no risk (stage 0), stage 1, and stage 2, respectively (Table 1). The average age was significantly higher in the L group (N: 53.42 ± 7.5, L: 56.89 ± 6.15, *p* < 0.001). Gender, height, weight, BMI, grip strength, and gait speed were not significantly different between the groups. There was no significant difference in the severity of LS between the N and L oxidative stress groups (stage 0: 41.7 and 42.5%, stage 1: 40.5 and 40.0%, and stage 2: 17.8 and 17.5% in the N and L groups, *p* = 0.90) (Table 2).

**Table 2.** The comparison of each parameter between the N and L groups in non-elderly participants.


BMI: body mass index; N group: participants with f(HMA) of 70% or more; L group: participants with f(HMA) less than 70%; LS: locomotive syndrome. Values are mean ± SD for each group.

#### *3.2. Elderly Participants*

The average age was 71.19 ± 5.24 years, and the mean f(HMA) was 67.09 ± 6.76%. Sixty-seven (36.8%) and 115 (63.2%) subjects were categorized in the N and L oxidative stress groups, respectively. In terms of LS, 66 (36.3%), 66 (36.3%), and 50 (27.4%) participants were grouped into stage 0, stage 1, and stage 2, respectively (Table 1).

Age and BMI were not significantly different between the N and L oxidative stress groups. There were significant differences in the percentage of LS in elderly participants (N: stage 0: 33 (49.3%), stage 1: 21 (31.3%), stage 2: 13 (19.4%); L: stage 0: 33 (28.7%), stage 1: 45 (39.1%), stage 2: 37 (32.2%); *p* = 0.004). There were significant differences in gender, height, weight, grip strength, and gait speed in elderly participants (*p* < 0.001, *p* < 0.001, *p* = 0.018, *p* < 0.001, *p* = 0.002, respectively) (Table 3).


**Table 3.** The comparison of each parameter between the N and L groups in elderly participants.

BMI: body mass index; N group: participants with f(HMA) of 70% or more; L group: participants with f(HMA) less than 70%; LS: locomotive syndrome. Values are mean ± SD for each group.

Since there were several factors with significant differences, they were examined as covariates for risk factors for elevated oxidative stress, as defined by f(HMA) > 70%: L group in logistic regression analysis, which found only LS as a risk factor for elevated oxidative stress (OR 0.515, 95% confidence interval, 95% CI: 0.281–0.943, *p* = 0.032) (Table 4). As the LS stage increased by 1, the risk of becoming L increased by 0.515 times.

**Table 4.** Logistic regression analysis for risk factors of the elevation of oxidative stress (L group) in elderly participants.


OR: odds ratio; CI: confidence interval; L group: participants with f(HMA) less than 70%; LS: locomotive syndrome.

#### **4. Discussion**

There have been several reports of the association between f(HMA), a marker of oxidative stress, and chronic diseases [16]. However, few reports have indicated its association with motor function [33]. Furthermore, to our knowledge, this is the first study to report on the association between f(HMA) and LS. The present study indicated that subjects with more severe LS stages had higher oxidative stress as assessed by f(HMA) levels in the elderly group, where oxidative stress was associated with a decline in locomotive function. The f(HMA) ratio could be a new biomarker associated with LS in elderly subjects.

Oxidative stress reflects the imbalance of reactive oxidative species and antioxidant defenses, and it plays an important role in the decline of body functions [7]. It has been reported that oxidative stress is associated with chronic diseases, including hypertension, obesity, liver injury, renal function, anemia, and cardiovascular complications [30]. Furthermore, increased oxidative stress in the elderly might be associated with a deterioration in motor function as increased oxidative stress reduces walking speed in elderly women [33]. This study concurred, showing increased oxidative stress and reduced motor functions, such as grip strength and walking speed, in elderly people, which worsened the degree of impairment of locomotion.

There are several possible mechanisms to explain the association between f(HMA) levels and motor performance in LS. Oxidative stress is associated with muscle function through several pathways, including changes in neuromuscular junctions, reduced muscle

energy metabolism, and reduced calcium release from the endoplasmic reticulum [11]. Another possible mechanism is muscle atrophy from increased proteolysis and decreased protein synthesis due to increased oxidative stress [11]. Oxidative stress has been reported to impair skeletal muscle as well as cardiovascular energy metabolism [11]. The association between f(HMA) and exercise capacity may be explained by the effect of oxidative stress on these systemic factors that determine exercise capacity.

In multivariate analysis, f(HMA) was associated with locomotion rather than simple motor functions, such as grip strength and gait, in the elderly group. LS is the concept of functional decline due to problems in bone, cartilage, muscle, and nerves [44], and f(HMA) may be related to abnormalities in these motor organs. However, an association between f(HMA) and decline in motor function or LS was not found in the non-elderly. The simple increase in oxidative stress does not affect motor function, but long-term exposure to oxidative stress, such as with age-related chronic inflammation, may be associated with a functional decline [45].

LS is a condition that requires nursing care. As it is a motor disease that is expected to improve with locomotion training, early detection leads to the preclusion of unnecessary nursing care [44]. In the current study, the oxidative stress marker f(HMA) was found to be associated with the degree of LS, and it may therefore be a new biomarker for the early detection of LS. If this is confirmed, it will be possible to intervene in LS from an early stage, resulting in nursing care being unnecessary. Furthermore, as exercise testing to diagnose LS is difficult in the limited time in an outpatient setting, if f(HMA) becomes a biomarker for suspected LS, it could provide a simple objective diagnostic modality for LS.

The modifiability of f(HMA) by intervention, and its responsiveness to changes in motor performance, need to be investigated in future studies. Supplementation with branched-chain amino acids is a potential intervention to increase f(HMA) levels. A previous study in patients with cirrhosis showed that administration of branched-chain amino acids increased f(HMA) [19]. LS may be improved by nutritional therapies that improve f(HMA), which may be a potential new treatment other than exercise therapy. There is scope to consider the link between LS, f(HMA), and nutritional status.

It may also be possible that the exercise regimens reported so far to improve LS may improve f(HMA) and reduce oxidative stress. Therefore, the results of this study may provide a basis for the hypothesis that exercise therapy, as previously described, improves systemic diseases caused by oxidative stress [46].

This study has several potential limitations. First, the participants were middle-aged and elderly people who lived in a relatively rural area, where many had jobs in agriculture or fishing. Thus, the lifestyle of these subjects differed from that of people in an urban environment. Furthermore, the participants attended for annual health examinations, which suggests that they may be more health conscious than other people. Second, this was a cross-sectional, single-center study. In the future, longitudinal and multicenter collaborative research will be needed to verify our findings. Finally, the specificity of f(HMA) as a biomarker of LS could not be discussed, because other biomarkers related to oxidative stress were not measured. Nevertheless, the present study still has a clinical application, by indicating that the redox state of HSA might serve as a biomarker for LS.
