The research protocol was approved at a meeting of the Ethics Commission of the L.T. Mala NIT NAMSU in accordance with the Declaration of Helsinki. All patients signed informed consent prior to any study-related procedures. The study group consisted of 88 apparently healthy subjects aged 47.6 [39.5; 54.7] 31-65 years old. There were 41 men (46.6 %) and 47 women (53.4 %) without established atherosclerotic cardiovascular disease (ACVD), metabolic disease (diabetes mellitus, obesity) or other chronic diseases who did not receive any drug therapy were screened. Exclusion criteria included a history of chronic disease, patient failure to follow study procedures, alcohol or drug abuse, and clinically significant laboratory abnormalities. Anthropometric studies were carried out according to standard methods. BMI was calculated using the formula body weight (kg) / height (m)². Body composition was determined by the bioelectrical impedance method using Composition Monitor BF511, Omron; body fat percentage (FAT, %), skeletal muscle percentage (MUS, %), and visceral fat level (VIS, %). Clinical blood analysis was performed on a MYTHIC18 automatic hematology analyzer. The content of insulin, C-reactive protein (CRP), tissue necrosis factor α (TNFα) and SIRT1 in blood serum was determined by the immunoenzymatic method using appropriate sets of reagents. The activity of total superoxide dismutase (T-SOD) in blood serum was determined by the colorimetric method. Blood lipid spectrum — total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) were determined by the enzymatic method using Cormay (Poland) reagent sets. The glycosylated hemoglobin content (%) (HbA1c) was determined via the photometric ion exchange method. The pro-oxidant/antioxidant balance of blood serum was calculated as the ratio of the content of total hydroperoxides (THP) and total antioxidant activity (TAA). Molecular genetic studies included the determination of the C/G polymorphism of the sirtuin gene (SIRT1, rs7069102), relative telomere length of blood leukocytes (RTL-b), and telomerase activity. Genotyping of the C/G polymorphic site of the sirtuin gene (SIRT1, rs7069102) was performed by real-time PCR. Telomere length was determined by real-time PCR10, 11. Amplification was performed using SsoAdvanced Universal SYBR Green Supermix (BioRad Laboratories, USA) and primer system (Thermo Fisher Scientific). Telomerase activity was determined by real-time PCR using the TRAPeze Kit RT Telomerase Detection Kit (Millipore, USA, CAT. No. S7710). Epigenetic age was calculated using the DNAm PhenoAge epigenetic clock based on nine biological markers: albumin (g/l); creatinine (mg/dL); glucose (mg/dL); CRP (mg/l); lymphocytes (%); Red Cell Distribution Width (RCDW) (%); Mean Cell Volume (MCV); alkaline phosphatase (U/l); leukocytes (x10⁹/l); age (years).

Continuous variables obtained from the participants in this study were expressed as means ± SDs. Categorical variables were expressed as percentages. Data were compared using the Mann-Whitney U test (for continuous variables) and the chi-square test (for categorical variables). To assess the interrelationship of indicators, a correlation analysis was performed with the calculation of pairwise correlation coefficients. Also, the associations between variables were assessed using linear multiple regression models. When estimating regression equations, the method of stepwise inclusion of predictors was used, which ranks features in accordance with their contribution to the model. The overall agreement between the model and the real data was evaluated using the Hosmer-Lemeshow Goodness-of-Fit Test. The Bonferroni test was used to correct for multiple comparisons for all analyses. P values < 0.05 were considered statistically significant. All analyses were conducted via the statistical package STATISTIСA V10.0.

Table 1

Characteristics	Groups		M-W p-value
	Accelerated ageing, n = 21	Healthy ageing, n = 67
HbA1c, %	5.96 ± 0.21	5.65 ± 0.48	0.007891
FPG, mmol/L	6.03 ± 0.67	5.23 ± 0.53	0.000001
Insulin, mMU/L	28.84 ± 11.46	20.07 ± 9.69	0.005124
HOMA IR	7.47 ± 3.06	4.76 ± 2.59	0.000849
TG, mmol/l	1.87 ± 0.78	1.36 ± 0.78	0.006248
TC, mmol/L	5.65 ± 1.45	5.64 ± 1.11	0.967165
LDL-C, mmol/L	3.64 ± 1.30	3.58 ± 0.97	0.934385
VLDL-C, mmol/L	0.92 ± 0.52	0.63 ± 0.36	0.009185
HDL-C, mmol/L	1.21 ± 0.26	1.41 ± 0.31	0.007328
Uric acid, µmol/L	5.65 ± 1.45	5.64 ± 1.11	0.967165
Albumin, g/L	339.18 ± 62.71	250.94 ± 64.89	0.000022
SIRT1, ng/ml	4.16 ± 1.18	3.47 ± 0.76	0.000662
TNFα, pg/ml	2.84 ± 1.37	2.36 ± 0.98	0.251498
T-SOD	49.75 ± 5.40	49.06 ± 4.15	0.432705
THP, µmol/l	153.32 ± 66.87	125.55 ± 44.88	0.693237
TAA, µmol trolox equivalent	484.92 ± 146.95	546.09 ± 160.27	0.176598
RTL-b, relative units	0.97 ± 0.28	1.00 ± 0.28	0.790882

Data are presented as the mean ± SD. P values were obtained using the independent-samples Mann–Whitney U test.

To determine the factors influencing aging rates, biological age (BA) was calculated using the DNAm PhenoAge epigenetic clock based on nine biological markers. After calculating BA, the rates of aging were determined for each examinee. If the patient's BA exceeded the calendar age by more than a year, the patient was assigned to the group of accelerated aging. If this was less than or equal to a year the patient was assigned to the group of healthy aging.

A comparative analysis of SIRT1 plasma levels revealed a difference between the accelerated and healthy aging groups. Accelerated aging was accompanied by a small but highly significant increase in SIRT1, which may indicate the activation of SIRT1-mediated metabolic pathways (Table 1). These groups also differed in a few biochemical indicators characterizing the state of carbohydrate and lipid metabolism, as well as the pro-antioxidant balance (Table 1).

The influence of SIRT1 gene polymorphisms has already been studied, but there is still no consensus on the association of various SIRT1 single nucleotide polymorphisms (SNPs) with the rate of aging and a predisposition to age-related diseases in humans. When choosing SIRT1 SNP, we proceeded from the fact that the results of the distribution of SNP polymorphisms rs2273773, rs3740051, and rs3758391 did not differ in mortality or age, as well as between frail and strong groups in senile frailty syndrome12, 13, 14, 15. Therefore, we decided to investigate SIRT1 SNP rs7069102 C>G polymorphisms located in intron 4, which elicited the most significant association with ACVD. SIRT1 SNP rs7069102 C>G was associated with an increased risk of subclinical coronary heart disease, an increase in the level of circulating SIRT1, and reduced expression of eNOS16, 17. The possible association of the SIRT1 SNP with a wide range of diseases and various pathological conditions may be consistent with the influence on the course of aging, which affects the susceptibility to many age-related diseases. The gene encoding SIRT1 may be closely associated with a risk of developing or the presence of diseases associated with metabolism, in particular, type 2 diabetes. This is because SIRT1 increases insulin secretion and fat deposition, affects insulin resistance, and is involved in regulating the metabolism of glucose and lipids by deacetylation of the histones of the corresponding transcription factors or target genes12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23.

Table 2

Genotype, allele	n = 88	%
С/С	5	5.68
C/G	45	51.13
G/G	38	43.18
C allele	55	31.25
G allele	121	68.75

Table 3

№	Genotype	N	SIRT1, ng/ml	Р (M-W)
1	С/С	5	3.37 ± 0.24	p1-2 = 0.741
2	C/G	44	3.62 ± 0.91	p1-3 = 0.969
3	G/G	37	3.57 ± 0.89	p2-3 = 0.842

Table 5

Parameters	R	p-value
LDL-C	-0.2235	0.039
Uric acid	0.2456	0.023
Albumin	-0.2931	0.006
TNFα	0.2616	0.014
Telomerase activity	-0.3860	0.001
THP	0.3251	0.003
TAA	-0.3494	0.001
THP / TAA	0.4750	0.001

Table 6

Parameters	b* ± Standard Error of b* Std.Err.[TMFSBTZ1] [ОЗ2]	b ± Standard Error of b	p-value
Intercept		6.372 ± 1.214	0.000001
THP / TAA	0.513 ± 0.080	1.764 ± 0.274	0.000000
Age	-0.158 ± 0.079	-0.011 ± 0.006	0.048
Uric acid	0.244 ± 0.088	0.003 ± 0.001	0.007
Monocytes	0.292 ± 0.082	0.123 ± 0.034	0.001
Albumin	-0.241 ± 0.083	-0.026 ± 0.009	0.005
RDW	-0.252 ± 0.079	-0.186 ± 0.058	0.002
Al AT	-0.239 ± 0.093	-0.009 ± 0.004	0.012
Leukocytes	0.179 ± 0.088	0.077 ± 0.038	0.044
BMI	-0.306 ± 0.128	-0.049 ± 0.021	0.019
Insulin	0.193 ± 0.100	0.013 ± 0.007	0.058
HbA1c	-0.161 ± 0.081	-0.310 ± 0.157	0.051
Waist circumference	0.243 ± 0.137	0.014 ± 0.008	0.079
RTL-b	0.108 ± 0.078	0.456 ± 0.331	0.173

When assessing the frequencies of G/G, C/G, and C/C genotypes of the SIRT1 SNP rs7069102 gene among individuals of the study group (Table 2), we found that results did not deviate from the Hardy-Weinberg equilibrium (p = 0.075).

The frequency of occurrence of the minor C allele was equal to 0.313 and did not significantly differ from the data of ALFA Allele Frequency (0.327) for the European population (https://www.ncbi.nlm.nih.gov/snp/rs7069102) and from the results for residents of Podillia, men without signs of cardiovascular pathology (0.350)24.

When analyzing the serum content of SIRT1 in carriers of different genotypes of the G/C polymorphism (rs7069102) of the SIRT1 gene, no significant differences between the study groups were found (Table 3).

In order to identify factors that may be associated with the presence of certain variants of the G/C polymorphism (rs7069102) of the SIRT1 gene, a comparative analysis of biochemical indicators was conducted in groups of patients carrying different genotypes of the specified polymorphism. Due to the low representation of C/C genotype carriers (n = 5), they were combined with C/G heterozygotes.

When conducting a comparative analysis of all defined indicators between groups, it was found that carriers of allele C had significantly higher activity of T-SOD (significance level p < 0.1) (p = 0.055) and shorter RTL-b (p = 0.102). No differences were found in the rest of the indicators.

It is known that the association of SIRT1 activity with life expectancy and SIRT1 expression declines with age. Therefore, we performed a comparative analysis of the serum level of SIRT1 in patients of different age groups. All patients were divided into groups depending on calendar age: group 1 — up to 45 years and below (n = 32), group 2 — from 45 years to 59 years (n = 52), and group 3 — over 60 years (n = 4). We found no significant differences in serum SIRT-1 between these groups (Table 4).

In order to measure the effect of SIRT1 on the regulation of metabolic pathways of aging, a correlation analysis was performed to determine the relationship between the serum level of SIRT1 and the primary metabolic and age parameters (Table 5).

Multivariate linear regression analysis was performed to establish both metabolic and molecular genetic factors associated with serum SIRT1 levels. (Table 6).

When conducting a dispersion analysis of the model and its significance, it was established that the contribution of the factors included in the model (Regress.= 28.56224) is 60.3% of the total sum of squared deviations of the predicted parameter (Total = 47.33818). In comparison, 39.7% of the contribution is due to random factors (Residual = 18.77595), which indicates the significance of the obtained model (F = 8.659218, p = 0.0000001).

According to recent studies, SIRT1 plays a crucial role in metabolism, immune response, oxidative status, and aging. SIRT1 regulates diverse cellular processes, including DNA repair, fat differentiation, glucose output, insulin sensitivity, fatty acid oxidation, neurogenesis, inflammation, and longevity25, 26, 27. It is also believed that SIRT1 may indirectly affect aging processes through the relationship between nutrition and aging10, 11, 12, 13. A study by Balestrieri et al. suggested that upregulation of SIRT1, which plays a key role as a metabolic sensor regulating glucose metabolism, may counteract the adverse effects associated with aging by reducing cardiovascular risk factors and28. In our study of apparently healthy subjects, we showed a significant association of SIRT1 with main metabolic factors, factors of pro-antioxidant balance and immune response: uric acid (R = 0.25; p = 0.023), TNFα (R = 0.26; p = 0.014), THP (R = 0.33; p = 0.003) and negative correlation with LDL-C (R = -0.22; p = 0.039), and TAA (R = -0.35; p = 0.001). The relationship between SIRT1 and telomerase activity was also confirmed (R = -0.39; p = 0.001).

Data on the effect of SIRT1 genotypes on SIRT-associated mechanisms of aging and on metabolic and age-related diseases, are contradictory29, 30, 31, 32, 33, 34, 35. Several studies have shown that various SIRT1 gene polymorphisms are associated with a predisposition to obesity32, arterial hypertension, and an increased risk of cardiovascular diseases, including carotid atherosclerosis33, 34, 35. Kilik et al. demonstrated a strong association between SNPs (rs7895833) of SIRT1 genotypes with lifespan: the oldest individuals (age ≥ 76.0 years) were carriers of AG genotypes for (rs7895833) and showed the highest SIRT1 levels and lifespan29. The results of the Rotterdam study showed an association of SIRT1 genotypes with BMI. In addition, Peters et al. showed that carriers of the rs7069102 G allele of the SIRT1 gene have a higher risk of obesity than carriers of the C allele variant32. However, our data did not confirm this association. The results of our study did not demonstrate an association of the SIRT1 genotype SNP (rs7069102) with either plasma SIRT1 levels, metabolic parameters, or chronological/epigenetic age. Some studies showed similar results and did not reveal a significant influence of the SIRT1 genotype. A study of centenarians (mean age 102.4 ± 2.3 years) conducted in China showed that eight common SIRT1 polymorphisms were not associated with human longevity. Meta-analysis of the role of SIRT1 (rs3758391) polymorphism, C vs T allele, did also not reveal a significant effect of these polymorphism alleles on life expectancy11, 30, 31. Such contradictory research results do not currently have a clear explanation. This may be due to the subjects included in the study. All these associations were found in individuals with a high risk of or existing chronic disease (ACVD, obesity, neurogenerative diseases), but were not confirmed in apparently healthy individuals. This may be why our study of apparently healthy individuals did not confirm that SNPs (rs7069102) of SIRT1 genotypes are associated with any metabolic parameters and the rate of aging. This issue, therefore, requires further study.

Experimental models have shown that SIRT1 may be involved in the mechanisms that inhibit the progression of atherosclerosis and provide protection against ACVD by inhibiting chemotaxis of mononuclear cells36, adhesion to the vascular wall37, formation of foam cells36, oxidative stress38 and anti-inflammatory factors39. At the same time, many questions remain regarding how to realize the protective and anti-aging effects of SIRT140, 41.

The attempts in clinical trials to interpret SIRT1 plasma levels in pathology compared with controls have been controversial: in some trials, SIRT1 plasma levels decreased, while in others, the levels increased compared with controls. For example, in the trial including obese patients, the level of SIRT1 was significantly lower (P = 0.002) in patients with obesity compared with lean controls42. Also, in the study by Esmayel I.M et al. (2021), the SIRT1 levels in patients with acute ischemic stroke were significantly lower than in the control group43. In contrast, opposite reports were observed in the other two trials. The SIRT1 level was significantly increased in the acute ischemic stroke group compared with the control group (0.63 ± 0.75 vs 0.48 ± 0.80 ng/mL; P ≤ 0.05) in the study by Liu Y et al.44. In the study by Liang X. et al., serum SIRT1 was significantly higher (P = 0.036) in acute ischemic stroke patients (0.62 ± 0.77 ng/mL) compared to healthy control subjects (0.45 ± 0.69 ng/mL), but SIRT1 concentrations were not significantly higher (0.58 ± 0.69 versus 0.64 ± 0.81 ng/mL, P = 0.298) than in patients in the unfavorable functional outcome group45.

Our results showed that in apparently healthy subjects, the SIRT1 level was higher in the accelerated aging group, in contrast to the healthy aging group (4.16 ± 1.18) ng/ml vs (3.47 ± 0.76) ng/ml, (p=0.00066), respectively.

Stepwise multivariate linear regression analysis shows the model (p=0.0000001) that supports multiple associations of SIRT1 with the primary metabolic processes in the development of aging and age-related disease (atherosclerosis, cardiovascular disease, hypertension, type 2 diabetes, cancer, and others).

SIRT1 plasma levels in apparently healthy subjects were associated with age, BMI, and WC, factors of carbohydrate metabolism, and markers of the pro-antioxidant balance. A comparative analysis of SIRT1 plasma levels between accelerated and healthy aging groups (by DNAm PhenoAge epigenetic clock) showed a significant difference. These groups also differed by metabolic parameters (HOMA IR, Hb1 AC, FPG, TG, VLDL-C, HDL-C). However, our study of apparently healthy subjects did not confirm that SNPs (rs7069102) of the SIRT1 genotypes are associated with SIRT1 plasma level, aging rate, or metabolic parameters.

ACVD: atherosclerotic cardiovascular disease; ALT: Alanine Aminotransferase; BA: biological age; BMI: index body mass; CAD: coronary artery disease; CRP: C-reactive protein; CVD: cardiovascular disease; HbA1c: glycosylated hemoglobin; HDL-C: cholesterol of high density lipoprotein; HOMA IR: Homeostatic Model Assessment of insulin resistance; FAT: body fat percentage; FPG: fasting plasma glucose; LDL-C: low-density lipoprotein cholesterol; MAD: metabolically associated disease; MCV: Mean Cell Volume; MUS: skeletal muscle percentage; RCDW: Red Cell Distribution Width; RTL-b: Relative telomere length of blood leukocytes; SD: standard deviation; SIRT1: sirtuin 1, SNP: single nucleotide polymorphism, TAA: total antioxidant activity; TC: total cholesterol; TG: triglycerides; THP: total hydroperoxides; TNFα: tissue necrosis factor α; T-SOD: total superoxide dismutase activity; VIS: visceral fat level; VLDL-C: very low-density lipoprotein cholesterol.

None.

Olena Kolesnikova: Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Olga Zaprovalna: Substantial contributions to the conception or design of the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Anastasiia Radchenko: the acquisition, analysis, or interpretation of data for the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Tetiana Bondar: Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

All authors read and approved the final manuscript.

None.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

This study was conducted in accordance with the amended Declaration of Helsinki. The institutional review board approved the study, and all participants provided written informed consent.

Not applicable.

The authors declare that they have no competing interests.