1. Introduction
Sarcopenia, defined as the age-related loss of muscle mass, strength, and physical performance, is a progressive, generalized musculoskeletal disorder that is associated with an increased risk of adverse events, such as falls, fractures, and impaired mobility [
1]. It is also associated with cardiac, respiratory, neurodegenerative, and cognitive complications that increase the need for nursing home care and reduce life expectancy [
2,
3,
4,
5]. As such, sarcopenia reflects senescence, a typical pathology associated with aging. Therefore, prevention, early diagnosis, and early therapeutic intervention will improve the quality of life and prognosis of elderly patients. However, diagnosing sarcopenia can be difficult because (1) multiple factors must be assessed, including muscle mass, strength, gait, and other physical functions, and (2) different reference values exist for men and women. Many older people will need care in the future, and older people with limited mobility are more likely to experience early onset frailty than healthy adults [
6]. Therefore, it is important to have a simple and accurate method for the prediction of sarcopenia risk.
In recent years, the relationship between blood polyamines and various health conditions has been noted [
7,
8,
9,
10,
11]. The natural polyamines spermine (SPM) and spermidine (SPD) and their precursor putrescine (PUT) are low molecular weight aliphatic amines that contain multiple amino groups. Polyamines, which are found in all organisms including humans, are essential for cell growth and differentiation and are involved in many physiological activities [
12]. Intracellular polyamine concentrations are altered by the activity of intracellular polyamine synthases and catabolites, as well as by polyamine supply from outside the cell and polyamine efflux from the cell. Enzymatic activities for polyamine synthesis decrease with age [
12], suggesting that polyamine levels decrease with age. In fact, when all age groups, including children, are examined, blood polyamine levels decrease with age [
13]. However, the age-related decline in tissue polyamine concentrations is observed only in early life (fetal and developmental periods) [
14]. No significant decrease in blood polyamine concentrations was observed in adults. However, several reports in humans have shown that the blood SPM concentration relative to blood SPD (SPM/SPD ratio) tends to decrease because SPM concentrations show a decreasing trend and SPD shows an increasing trend [
15,
16,
17,
18].
Blood polyamine levels are influenced by the supply of polyamines from outside the cells. For example, we have previously reported that a polyamine-rich diet increases blood levels of SPM, which has much stronger biological activities than SPD. This increase was accompanied by evidence of SPM bioactivity, such as suppression of age-related pro-inflammatory conditions in mice and humans [
15,
19]. In addition, we have shown that a long-term increase in polyamine intake prolongs the life span of mice [
20]. We also found that SPM activates DNA methyltransferases and inhibits the progression of DNA methylation abnormalities associated with polyamine deficiency and aging [
21,
22]. The increase in blood SPM concentration and the concomitant increase in the SPM/SPD ratio were associated with a positive health status, which subsequently contributed to an increased healthy life expectancy.
Conversely, blood levels of SPD are elevated in patients with several age-related conditions, including impaired cognitive function [
7,
9] and neurodegenerative diseases such as Alzheimer’s disease [
10] and Parkinson’s disease [
11]. In fact, the age-related decline in the SPM/SPD ratio is accelerated in these patients [
8]. The mechanism of the increase in SPD levels and the decrease in SPM/SPD ratio is not well understood; however, it is necessary to consider that an increase in the SPD levels is a complementary mechanism that improves the disease state by stimulating autophagy activation in the brain. One reason for this is that the basic properties of polyamines were not understood in the experiments investigating the activation of autophagy by SPD, and thus artifacts other than the bioactivity of SPD itself were considered bioactive [
23]. In addition, the blood–brain barrier prevents polyamines from entering brain tissue [
12]. Therefore, we speculate that increased SPD concentrations and a decreased SPM/SPD ratio may lead to the development and progression of lifestyle-related diseases that worsen with age.
Sarcopenia is a typical condition that develops and progresses with age. While previous research has shown that blood polyamines are associated with age-related diseases, no previous study has examined whether they are associated with the typical age-related pathological condition, sarcopenia. In addition, a simple diagnostic approach to predict the risk of developing sarcopenia has not yet been developed. Therefore, in this study, we investigated whether blood polyamine levels are associated with sarcopenia and whether blood polyamine levels can be used as a biomarker for the early detection or assessment of progression of sarcopenia.
2. Materials and Methods
2.1. Study Design and Participants
In this cross-sectional study, we included elderly persons aged 70 years or older who visited outpatient clinics affiliated with Minamiuonuma City Hospital (Minamiuonuma City, Japan) or who resided in nursing homes for the elderly (Minami-en and Maiko-en, Minamiuonuma, Japan). A flowchart of the patient selection process is shown in
Figure 1. Patients with pacemakers were excluded because bioelectrical impedance analysis (BIA) cannot be used to measure muscle mass in this population. Since neoplasms have a significant impact on polyamine levels, patients with cancer and those with a history of cancer treatment within 3 months were excluded.
2.2. Data and Sample Collection
Medical history and clinical information such as age, history of cardiovascular disease (CVD) and cerebrovascular disease (CeVD), history of dementia and fractures were obtained from medical records, physical examination results, and questionnaires. Height and weight were measured in the standing position. However, the height of participants who were unable to stand was measured with a tape measure. Measurements were taken from the top of the head to the heel. The weight of the participants who were unable to stand was calculated by subtracting the weight of the wheelchair from the value measured in the wheelchair. Body mass index (BMI) was calculated as weight (kilograms) divided by height (meters squared). For the measurement of body composition and muscle mass, a multi-frequency BIA was performed using the InBody S10 (InBody Japan, Tokyo, Japan). The skeletal muscle mass index (SMI) was calculated by dividing the skeletal muscle mass of the limbs in kilograms by the square of the height in meters. Hand grip strength (HGS) was measured with a hand grip strength meter (Smedley’s Hand Dynamometer; Matsumiya Ika Seiki, Tokyo, Japan) while the patient was seated, and the higher value of the left- and right-hand grip strength tests was used. The walking speed (WS) was calculated as the time required to walk 10 m at a self-selected WS.
2.3. Definition of Sarcopenia
We defined sarcopenia as a decrease in muscle mass and a decrease in muscle strength or physical performance according to the diagnostic criteria established by the Asian Working Group for Sarcopenia (2019) [
1]. Thus, a diagnosis of sarcopenia was made based on the following: SMI < 7.0 kg/m
2 and <5.7 kg/m
2 for men and women, respectively, plus either an HGS value of <28 kg and <18 kg for men and women, respectively, or WS less than 1.0 m/s. Participants who did not meet the criteria for sarcopenia were assigned to the non-sarcopenia group. For further analysis, the non-sarcopenia group was subdivided as follows: a semi-sarcopenia group (patients who had either a decrease in SMI or a decrease in HGS and/or WS but who did not meet the criteria for a sarcopenia diagnosis) and a healthy group (the remaining patients from the non-sarcopenia group).
2.4. Blood and Biochemical Tests
Blood samples were collected from participants at room temperature. Whole blood samples for measurement of polyamine concentrations were immediately stored at −20 °C until assayed. All measurements of blood samples, except for polyamines, were performed in a clinical laboratory at Minamiuonuma City Hospital. Hemoglobin concentration (g/dL) was measured via the sodium lauryl sulfate hemoglobin detection method using an automated blood cell analyzer (XN-2000; Sysmex Corporation, Kobe, Japan). An automated biochemical analyzer (BM6070; JEOL Ltd., Akishima, Japan) was used to perform biochemical blood tests. The concentration of albumin (Alb) in serum (g/dL) was measured using the modified bromocresol purple method. Creatinine levels were measured using the enzymatic method, and the estimated glomerular filtration rate (eGFR; mL/min/1.73 m2) was calculated using the following formulas: eGFR (mL/min/1.73 m2) = 194 × creatinine −1.094 × age −0.287 (for men); eGFR (mL/min/1.73 m2) = 194 × creatinine −1.094 × age −0.287 × 0.739 (for women). Low-density lipoprotein cholesterol (LDL-C) (mg/dL) and high-density lipoprotein cholesterol (mg/dL) levels were measured via direct assay. Triglycerides (TG) (mg/dL) were measured using the enzymatic method. Hemoglobin A1c (HbA1c) (%) was measured using high-performance liquid chromatography (HPLC) (AH8290; ARCRAY Inc., Kyoto, Japan).
2.5. Determination of Polyamine Concentrations in Whole Blood
Whole blood samples were heparinized, collected, and stored at −20 °C or below. Whole blood was thawed and degraded using sonication and freeze–thaw cycles to measure polyamine concentrations. Polyamine concentrations were determined via HPLC [
24] at the Cardiovascular Institute for Medical Research, Saitama Medical Center, Jichi Medical University. For polyamine extraction, whole blood was diluted fivefold with 5% trichloroacetic acid (TCA) and incubated at 95 °C for 45 min. After centrifugation at 13,000×
g for 20 min at 4 °C, the supernatant was collected and deproteinized by increasing the TCA concentration to 10% and incubating at 95 °C for 45 min, followed by centrifugation at 13,000×
g for 20 min at 4 °C. Polyamines in 20 µL of the TCA supernatant were separated with an HPLC system (Shimadzu Corporation, Kyoto, Japan) using a TSKgel Polyaminepak column (column size 4.6 mm ID × 50 mm length, particle size 7 µm, TOSOH Bioscience, Tokyo, Japan) at 50 °C. The flow rate was set at 0.42 mL/min and the composition of the separation buffer adjusted to pH 5.10 was 0.09 M citric acid (NACALAI TESQUE, INC., Kyoto, Japan), 2 M NaCl (NACALAI TESQUE, INC., Kyoto, Japan), 0.64 mM n-capric acid (NACALAI TESQUE, INC., Kyoto, Japan), 0.1% Brij-35 (Sigma-Aldrich Japan, Tokyo, Japan), 20% methanol (FUJIFILM Corporation, Osaka, Japan). Polyamines were detected via fluorescence intensity after the column effluent was reacted with the solution containing 0.4 M boric buffer (pH 10.4) (NACALAI TESQUE, INC., Kyoto, Japan), 0.1% Brij-35, 2.0 mL/L 2-mercaptoethanol (NACALAI TESQUE, INC., Kyoto, Japan), and 0.06% o-phthalaldehyde (NACALAI TESQUE, INC., Kyoto, Japan) at 50 °C. The flow rate of o-phthalaldehyde solution was 0.42 mL/min and fluorescence was measured at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The retention time was 12 min for SPD and 23 min for SPM. Concentrations in the original whole blood samples are expressed in micromoles.
Blood SPD, SPM, and SPM/SPD ratio were each measured three consecutive times in two samples and the average was calculated as the %CV (coefficient of variation). The %CVs were 4.3% for SPD, 3.6% for SPM, and 6.7% for the SPM/SPD ratio.
2.6. Statistical Analysis
The Shapiro–Wilk test was used to determine whether the variables were normally distributed. Data are presented as mean ± standard deviation for normally distributed data, and median and interquartile range for non-normally distributed data. To evaluate differences in variables between the sarcopenia and non-sarcopenia groups, normally distributed variables were evaluated using the unpaired Student’s t-test, and non-normally distributed variables were evaluated using the Mann–Whitney U test.
Comparisons between the three groups were made as follows. The z-test was used for multiple comparisons of proportions, and the Bonferroni method was used to determine significance. Specifically, the Bonferroni method was used to obtain the “adjusted significance level (p′)”, and a judgment was made at p′ = 0.017 for the probability value of the significance test (z-test) results for each pair of comparisons. Results of the comparison between groups were considered significantly different at p < 0.05. The Kruskal–Wallis test was used for multiple comparisons of values. Levene’s test was used to assess homogeneity of variance. Tukey’s method was used to determine between-group differences for variables with equal variance, and Games-Howell’s method was used for variables with unequal variance.
Correlations between age and SPD, SPM, or the SPM/SPD ratio were evaluated by using Spearman’s rank correlation coefficient. Multivariate logistic regression models were used to calculate adjusted odds ratios (ORs) with 95% confidence intervals (CIs) for the risk of sarcopenia associated with the SPD and SPM concentrations or the SPM/SPD ratio. Because SMI, HGS, and WS are diagnostic criteria for sarcopenia and nursing home occupancy rates reflect the consequences of developing of sarcopenia, analyses were performed after excluding these factors.
All analyses were performed using IBM SPSS Statistics for Windows version 28.0 (IBM, Armonk, NY, USA). A two-tailed p-value < 0.05 was considered statistically significant.
4. Discussion
Previous studies of age-related changes in blood polyamine concentrations in individuals under the age of 80 have shown a decreasing trend in SPM/SPD, although the difference is not statistically significant [
15,
16,
17,
18]. In this study, we observed a tendency for the SPM/SPD ratio to decrease with age in the sarcopenia group; this tendency was not observed in the non-sarcopenia group. Additionally, the data showed that the SPD concentration was significantly higher and the SPM/SPD ratio was significantly lower in patients with sarcopenia than in those without sarcopenia. Elevated blood levels of SPD have been reported in patients with impaired cognitive function [
7,
9] and several neurodegenerative diseases [
10,
11]. In addition, a decreased SPM/SPD ratio has been reported in neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease [
8].
Blood polyamine levels reflect systemic polyamine concentrations. SPD has been found to be present at higher concentrations than SPM in many mammalian organs and tissues, as reflected by the low SPM/SPD ratio in mouse tissues [
25]. However, in the same study, the concentration of SPM was comparable to that of SPD in several murine organs. In addition, SPM/SPD ratios were higher in the brain and muscle than in other organs. Although the blood–brain barrier is thought to block the entry of polyamines from the blood into the brain, changes in polyamine concentrations in the brain are thought to be reflected in blood polyamine concentrations via the spinal fluid. Sarcopenia has been associated with decreased muscle mass and brain volume in humans [
26]. This implies cell disintegration, and the polyamines released from within the cell as a result would be reflected in the blood levels. Although the SPM/SPD ratio should increase as polyamines leak from brain and muscle cells where SPM is abundant, our data show progressively lower SPM/SPD ratios in the following order: healthy, semi-sarcopenia, and sarcopenia. This finding suggests that changes in polyamine levels in patients with sarcopenia are not simply caused by cell destruction.
Inflammation, which is involved in the onset and progression of senescence and age-related pathologies, activates enzymes such as spermidine/spermine
N1-acetyltransferase (SSAT) and spermine oxidase (SMO) [
27,
28]. SSAT, together with acetylpolyamine oxidase (AcPAO), converts SPM to SPD and simultaneously converts SPD to PUT. On the other hand, SMO primarily converts SPM to SPD but does not act on SPD (
Figure 4). Overall, the induction of inflammation causes more SPM to be degraded. In contrast with non-inflammatory conditions, inflammatory conditions result in significantly greater SPM degradation. This may be the mechanism responsible for the increased SPD concentration and decreased SPM/SPD ratio observed in the sarcopenia group. It is also important to note that neurodegenerative diseases that have been linked to polyamines have also been found to be closely associated with chronic inflammation [
29,
30,
31]. Additionally, patients with these diseases have been found to have elevated blood SPD levels and decreased SPM/SPD ratios [
7,
9,
10,
11].
Again, the increase in SPD was unlikely to be due to a compensatory increase in absorption from the gastrointestinal tract, as there were no specific dietary or other interventions for patients with sarcopenia. Even if we assume that elevated SPD exerts a feedback function to promote physiological activity in patients with sarcopenia, there is no scientific evidence to explain this. First, there is little evidence of reduced SPD in tissues and organs of neurodegenerative diseases and similar age-related diseases. Second, it has been shown that a diet high in spermidine does not lead to an increase in spermidine concentrations [
7,
15,
21]. Third, because the blood–brain barrier prevents polyamines in the blood from entering the brain. Even if blood concentrations increase by a feedback mechanism due to a decrease in brain concentrations, polyamines outside the brain will not affect polyamine concentrations in the brain and will not exert any biological activity.
AcPAO oxidizes acetylated polyamines converted through SSAT, producing H
2O
2 and 3-acetoamidopropanal (3-AAP) as by-products. SMO directly converts SPM to SPD to produce H
2O
2 and the aldehyde 3-aminopropanal (3-AP) (
Figure 4). The produced 3-AP is spontaneously deaminated to produce acrolein, a highly toxic aldehyde [
32]; however, little acrolein is produced from 3-AAP [
33]. It has been reported that acrolein and 3-AP are highly cytotoxic, whereas 3-AAP is not [
34]. Findings suggesting inflammation-induced activation of SMO to degrade SPM and produce acrolein include increased acrolein levels in renal failure [
35], cerebrovascular disease [
36], and other age-related conditions that have been reported to have adverse health effects [
37]. In addition to inflammation-induced oxidative stress, the cytotoxic activities of 3-AP and acrolein produced by SMO may play an important role in the progression of sarcopenia and many other age-related diseases.
There are several reasons why we measured polyamine concentrations in whole blood rather than in serum or plasma. First, it can be very difficult to accurately detect polyamines, especially SPM levels, in the serum or plasma. This is because most polyamines in the blood are found in blood cells [
38]. Accurate measurement of SPM concentration at low levels can be very difficult because HPLC cannot distinguish between SPM peaks and noise that occurs as baseline variation [
39]. The second is to eliminate the effect of hemolysis on serum and plasma polyamine levels. When hemolysis occurs in a blood sample, it releases polyamines, which are present in large quantities in blood cells, and even a very small amount can have a significant effect on polyamine concentrations. Measuring polyamine levels in whole blood requires special techniques, but we have established a method to ensure accurate detection.
Due to the large inter-individual variability in polyamine concentrations [
12], it may be difficult to identify a cut-off value at which sarcopenia risk can be determined with a single measurement. Additionally, because various factors related to the conditions under which polyamine concentrations are measured can cause differences in measured polyamine concentrations, we believe it is difficult to compare and evaluate values measured at different times and to track progress. However, the SPM/SPD ratio can be measured as a relative value with a single measurement. Therefore, the data are reliable for individual and long-term comparisons, even if absolute polyamine concentrations vary due to differences in measurement methods. As a result, we believe that observing the time-dependent changes in the SPM/SPD ratio, which decreases as sarcopenia progresses, can be used to determine the risk of developing sarcopenia.
We found that the SPD concentration was significantly higher and the SPM/SPD ratio was significantly lower in patients with sarcopenia than in those without sarcopenia, and these levels were also found to change with the severity of sarcopenia. Due to the large individual variability in polyamine levels, it is difficult to identify a cut-off value at which the risk of developing sarcopenia can be determined. However, in conclusion, we believe that the risk of developing sarcopenia can be determined by observing changes in the blood SPM/SPD ratio over time, which is a relative value.