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Sobenin et al. Vessel Plus 2019;3:14 I http://dx.doi.org/10.20517/2574-1209.2018.63 Page 3 of 10

METHODS
Patients
This study was kept in accordance with the Helsinki Declaration of 1975 as revised in 1983, 2008 and 2013,
and was approved by the local ethics committee. The study participants were recruited from the visitors’
flow at municipal outpatient clinics, who have passed a routine screening for cardiovascular risk factors. In
total, 220 study participants were recruited (97 men, 123 women) with a mean age of 65.1 years (SD 9.4). The
written informed consent was obtained from all participants prior to inclusion in the study.

MetS diagnostics
[10]
For identification of patients with clustered risk factors and MetS, IDF 2009 criteria were used . In brief,
waist circumference > 94 cm in men and > 88 cm in women, triglycerides ≥ 150 mg/dL or drug treatment for
elevated triglycerides, HDL cholesterol ≤ 40 mg/dL in men and ≤ 50 mg/dL in women, or drug treatment for
reduced HDL cholesterol, systolic blood pressure ≥ 150 mmHg and/or diastolic blood pressure ≥ 85 mmHg,
or antihypertensive drug treatment, and fasting glucose ≥ 5.6 mmol/L or drug treatment of elevated glucose
were taken in consideration. The presence of at least 3 of above criteria was required for the diagnosis of
MetS. The group of MetS-free study participants consisted of subjects who had 0-1 of the above criteria. The
subjects with 2 MetS criteria were not eligible for inclusion in the study to avoid possible uncertainties. To
formally describe the severity of MetS (the extent of clusterization of conventional risk factors), an unofficial
integral MetS index was calculated as a simple multiplication of the absolute values of waist circumference,
triglycerides, systolic and diastolic blood pressure, and fasting glucose, divided by HDL cholesterol.

Ultrasonographic examination
For diagnostics of carotid atherosclerosis, high-resolution B-mode carotid arterial ultrasonography
imaging was used (SSI-6000 ultrasound system, SonoScape, China, equipped by 7.5-MHz L741 linear array
[21]
probe). The protocol of ultrasound examination developed earlier by Salonen et al. , 1995 was used. The
cIMT measurements were carried out with M’Ath software package (IMT, France). The extent of carotid
atherosclerosis and the size of atherosclerotic plaques were evaluated as described elsewhere [17,22] .

MtDNA genotyping
DNA was isolated from circulating leukocytes (whole venous blood) using a commercial kit for DNA
isolation and purification (QIAGEN GmbH, Germany) according to manufacturer’s instructions. DNA
concentration in samples was determined by NanoPhotometer Pearl UV/Vis SDRAM P-34 (IMPLEN,
Germany); the samples were kept in TE buffer at a concentration of 0.03 µg/µL. Heteroplasmy levels of
mtDNA mutations m.652delG, m.1555A>G, m.3336T>C, m.3256C>T, m.5178C>A, m.12315G>A, m.13513G>A,
m.14459G>A, m.14846G>A, and m.15059G>A were analyzed on Real Time PCR System 7500 Fast (Applied
Biosystems, USA) by qPCR (5’-terminal tag excision, TaqMan Assay). The nucleotide sequences for primers
and probes are shown in Table 1.

For qPCR, 4 µL DNA was added to 25 µL of standard reaction mixture [1x TaqMan Buffer, 3 mmol/L MgCl ,
2
250 µmol/L of each dNTP, 300 nmol/L primers, 300 nmol/L hybridization probes, 0.5 units Taq-polymerase
(Helicon, Moscow, Russia)]. Denaturation was held for 2 min at 94 ˚C; amplification stage with fluorescence
detection included denaturation for 10 s at 94 ˚C, and annealing for 15 s at a temperature specified for each
pair of primers and probes (range 61 ˚C-67 ˚C).

Statistical analysis
The statistical analysis was done using the IBM SPSS 22.0 software package (IBM Corp., Chicago, IL, USA).
The methods of descriptive statistics, correlation analysis by Spearman and Pearson, and one-way analysis
of variance (ANOVA) were used. Mean values were compared by T-test or U-test by Mann-Whitney for
continuous variables, and χ Pearson’s test for categorical variables. To assess the homogeneity of variance,
2