**1. Introduction**

The majority of developed countries have an aging population [1], and the number of people requiring support and care in their daily lives due to musculoskeletal disorders is increasing [2]. Locomotive syndrome (LS), which is a condition of reduced mobility due to impairment of locomotive organs, was proposed by the Japanese Orthopedic Association (JOA) as an overarching term for this condition [2,3]. LS has received worldwide attention for an assessment of the motor function in motor diseases [4]. LS is associated with a significantly lower quality of life (QOL) [5] and a shorter life expectancy. Prevention of LS has long been advocated for maintaining and improving physical function in middle-aged and elderly people [6].

**Citation:** Ito, S.; Nakashima, H.; Ando, K.; Kobayashi, K.; Machino, M.; Seki, T.; Ishizuka, S.; Kanbara, S.; Inoue, T.; Koshimizu, H.; et al. Human Nonmercaptalbumin Is a New Biomarker of Motor Function. *J. Clin. Med.* **2021**, *10*, 2464. https:// doi.org/10.3390/jcm10112464

Academic Editor: Johannes C. Reichert

Received: 24 April 2021 Accepted: 31 May 2021 Published: 2 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Oxidative stress reflects the imbalance of reactive oxidative species and antioxidant defenses and plays an important role in the decline of body functions in old age [7–9]. Elevated oxidative stress induces apoptosis of skeletal muscle [10], abnormality in neuromuscular junctions [11] and impaired mitochondrial function [12], resulting in decreased muscle performance, one of the major determinants of exercise capacity [13]. A recent systematic review of older adults has shown an association between increased oxidative stress and physical frailty [14]. Given that oxidative stress is one of the origins of agerelated decline in functional reserve, the use of biomarkers that reflect the redox status of the body may allow early identification of individuals at risk of functional decline due to musculoskeletal disease. The human serum albumin (HSA) cysteine-34 accounts for about 80% of the extracellular free thiols and is a major extracellular antioxidant [15]. Thus, HSA has been considered an important scavenger of reactive oxidative species, for example hydroxyl radical and hydrogen peroxide [16], but there are reports of differing antioxidative effects of HSA depending on its chemical structure. For example, Cys-34 residue functions as a universal antioxidant residue with excellent scavenging ability against a variety of reactive oxygen species, while Met residue may play an auxiliary role [17,18].

HSAs have been chemically classified into two major categories based on their redox status: human non-mercaptalbumin (HNA: oxidized form) and human mercaptalbumin (HMA: reduced form) [19]. Under oxidative stress, HMA changes to reversibly oxidized HNA-1 and highly oxidized HNA-2. Under oxidative stress, HMA buffers reactive oxidized species and turns them into HNAs; therefore, the proportion of HMA in HSA (f(HMA)) has been considered a biomarker reflecting the redox status of the human body [20]. Although the proportion of each HSA form is generally age- and disease-dependent, studies have shown that HMA, HNA-1, and HNA-2 account for 70–80%, 20–30%, and 2–5%, respectively, of the total albumin in healthy young adults [21].

Several clinical studies have examined the relationship between the redox status of HSA and the severity and progression of hypertension [22,23], obesity [24], liver injury [25], renal function [26,27], anemia [28], and cardiovascular complications in patients on dialysis [29,30]. It is also associated with Diabetes Mellitus [31] and Alzheimer's disease [32].

Although limited epidemiological studies have analyzed the association between HSA redox status and motor function [33], this redox status might be a biomarker for LS. The purpose of this study was to evaluate the redox status of albumin in a middle-aged and elderly Japanese population, and to investigate its correlation with motor function, including LS.

#### **2. Materials and Methods**

#### *2.1. Study Participants*

The individuals surveyed were volunteers who underwent a municipal-supported health checkup in the town of Yakumo in 2016. The town of Yakumo has a population of about 17,000 of whom 28% are over 65 years old. More people are engaged in agriculture and fishing than in urban areas. This town has been conducting annual health checkups since 1982. Physical examinations include voluntary orthopedic and physical function tests, internal examinations, and psychological examinations, as well as a health-related QOL survey (SF-36) [34,35]. This study included all participants who completed an assessment of the LS risk stage. The exclusion criteria were: a history of spine or joint surgery, severe knee injury, severe hip osteoarthritis, history of hip or spine fractures, neuropathy, severe mental illness, diabetes, kidney or heart disease, non-fasting, severe impairment of walking or standing, and impairment of the central or peripheral nervous system.

Of the 555 participants who underwent health checks, 306 (128 men and 178 women) met the inclusion criteria. The research protocol was approved by the Human Research Ethics Committee and the University's Institutional Review Board (No. 2014-0207). All participants gave written informed consent prior to participation. The research procedure was carried out in accordance with the principles of the Declaration of Helsinki.

#### *2.2. Examination of Motor Function*

Grip strength in the standing position was measured once for each hand with a handgrip dynamometer (Toei Light Co., Ltd., Saitama, Japan), and the mean value was used [36]. Subjects walked a straight 10 m course once at their fastest pace, and the time required to complete the course was recorded as the 10 m gait time [37].

#### *2.3. LS Stage Tests*

To evaluate the risk of LS, the JOA has proposed three tests: the two-step test, the stand-up test, and the 25-question geriatric locomotive function scale (GLFS-25) [2]. LS is categorized into stages 1 and 2, and these tests assess the degree of motor function and define the stages of LS. Stage 1 indicates that movement function has begun to decline, and stage 2 indicates that movement function has progressed towards a decline in mobility.

Three tests were conducted according to the JOA guidelines [2].

In the stand-up test, the ability to stand with a single- or double-leg stance from stools of heights, 40, 30, 20, and 10 cm, is evaluated. The grading of difficulty, from easy to difficult, is in the order of double-leg stance with 40, 30, 20, and 10 cm stools, followed by single-leg stance with 40, 30, 20, and 10 cm. The test result is expressed as the minimum height of the stool that the subject was able to stand up from.

In the two-step test, a physical therapist measured the length of two steps from the starting line to the tip of the toe. Scores were calculated by normalizing the maximum length of two steps by height.

The GLFS-25 is a self-reported comprehensive survey that refers to the previous month [38]. The scale consists of four questions about pain, 16 questions about Activities of Daily Living (ADL), three questions about social functioning, and two questions about mental status. Each item was graded from no disability (0 points) to severe disability (4 points).

We defined LS0, 1, 2 as follows:

LS0

> The subject is categorized as Stage 0 if all three of the conditions are met as follows:


LS1

> The subject is categorized as Stage 1 if any of the three conditions are met as follows:


The subject is categorized as Stage 2 if any of the three conditions are met as follows:


#### *2.4. Measurements of HSA*

During the checkup, fasting blood samples were collected through venipuncture and centrifuged within 1 h of sampling. Serum samples were stored at −80 ◦C until the assay was performed. Routine biochemical analyses were performed in the laboratory of the Yakumo Town Hospital. Interpersonal measurements of height and weight were taken to calculate the body mass index (BMI, kg/m2).

The determination of HSA, HNA, and HMA using high performance liquid chromatography (HPLC) with an ultraviolet detector has been reported by Sogami et al. [39]. In this study, the HPLC-post-column bromocresol green (BCG) method was used, which was

engineered to ensure that serum uric acid and bilirubin did not interfere with chromatographic peaks [40]. Frozen serum samples were thawed at room temperature and filtered through a Mini-UniPrep syringe-less filter (Agilent, Tokyo, Japan); HPLC was performed and reacted with BCG reagents to separate HMA and HNA detected at a 620 nm wavelength. The sample volume injected into the HPLC was 5 μL. The mobile phase reagen<sup>t</sup> consisted of *N*-methylpiperazine-HCl buffer (pH 4.5), 40 mM Na2SO4, and 3% ethanol; the BCG reagen<sup>t</sup> consisted of 150 mM citric acid, 3% Brigi 35, and 0.3 mM BCG. For all experiments, distilled water deionized to 18 m Ω using the Millipore Milli-Q System (Millipore Co., Bedford, MA, USA) was used.

The HPLC system used in this study was the Hitachi Lacrom Ice System (Hitachi, Tokyo, Japan), which consisted of an isocratic pump (L-2130), an auto-injector (L-2200), and a column oven (L-2300). Chromatograms were obtained using a photodiode array detection system (model L-2455). A Shodex Asahipak GS-570 GS column (100 mm × 7.5 mm ID) was used to separate the HSA components before sample injection.

In the present experiment, the peak of HNA-2 was not sufficiently quantified, and its peak area was not considered in subsequent analyses. To numerically assess the redox state of HSA from the HPLC profile, f(HMA), which represents the ratio of the peak area of HMA to the peak area of HSA, has been used in previous similar studies [41]. Hence, we followed these reports for the present study.

The f(HMA) was calculated using the following equation: f(HMA) = HMA area/(HMA area + HNA area) × 100.

Previous studies have demonstrated that f(HMA) accounts for 70–80% of the total albumin in a healthy young adult [42]. Therefore, the cut-off value of f(HMA) was determined to be 70%. We divided the participants into the normal (N, f(HMA) ≥ 70%) and lower (L, f(HMA) < 70%) oxidative stress groups.

#### *2.5. Statistical Analysis*

Continuous variables were expressed as mean ± standard deviation (SD). We compared continuous variables of the L group to those of the N group using the student t-test, and categorical variables of the L group to those of the N group using the Chi-squared test. Logistic regression analysis was performed to evaluate important risk factors of elevated oxidative stress, as defined by f(HMA) < 70%: L group. The dependent variable was N versus L groups. Following univariable analysis, variables that yielded a *p*-value < 0.20 were included in the multivariable analysis.

Each analysis was done separately for the under-65 (non-elderly) and the over-65 (elderly) groups.

All statistical analyses were performed using SPSS Statistics v.22.0 software for Mac (IBM Corp., Armonk, NY, USA). A *p*-value < 0.05 was considered significant in all analyses.
