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Sobenin et al. Vessel Plus 2019;3:15 I http://dx.doi.org/10.20517/2574-1209.2019.09 Page 3 of 9
hypertension (diastolic blood pressure > 90 mmHg and/or systolic blood pressure > 140 mmHg, or prescribed
to antihypertensive drugs), the presence of diabetes mellitus or impaired glucose tolerance (blood sugar >
110 mg/dL, or prescribed to regular insulin injections and/or sugar-lowering drugs), smoking (consumption
of > 10 cigarettes per day for thirty preceding months), and the presence of family history of cardiovascular
diseases. For women, an additional inclusion criterion was the presence of menopause (spontaneous or
surgical) of more than 5-years duration.

Exclusion criteria were: abnormal anatomical configuration of the neck and neck muscles, severe
tortuosity or unusual layout of the carotid arteries and its branches, the history of clinical manifestations
of atherosclerosis (myocardial infarction, angina pectoris, acute and transient cerebral ischemic attacks,
aortic aneurisms, revascularization of carotid, coronary or peripheral arteries), chronic heart failure of 3-4
functional class, and the presence of severe concomitant diseases.

Clinical examination of study participants included a biochemical analysis of serum lipid profile,
identification of the main cardiovascular risk factors, calculating a prognostic 10-year risk of developing
[6]
coronary heart disease, myocardial infarction and sudden coronary death , and quantitative ultrasound
diagnostics of carotid atherosclerosis.

Lipid analysis
Venous blood was taken after overnight fasting. To obtain serum, the blood was incubated for 1 h at 37 °C and
centrifuged for 15 min at 1,500 g. Cholesterol and triglyceride levels were measured by commercial enzymatic
kits (Cholesterol-32-Vital, and Triglycerides-22-Vital, respectively; Vital Diagnostics SPb, St. Petersburg,
Russia). Serum HDL cholesterol concentrations were measured after precipitation with magnesium chloride
phosphotungstic acid reagent (HDL-Cholesterol-04-Vital, Vital Diagnostics SPb, St. Petersburg, Russia).
Serum LDL cholesterol was calculated by Friedewald formula as the difference between total cholesterol and
the sum of HDL cholesterol and 1/5 triglycerides, and the ratio LDL-C/HDL-C was calculated.

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
[7]
probe). The protocol of ultrasound examination developed earlier by Salonen et al. , was used. The cIMT
measurements were carried out by a certified operator with M’Ath software package (IMT, France). The
extent of carotid atherosclerosis were evaluated as described elsewhere .
[8,9]

MtDNA genotyping
DNA was isolated from whole venous blood with commercial kit for DNA isolation and purification
(QIAGEN GmbH, Germany). 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.
Genotyping of mtDNA was performed for heteroplasmic mutations m.652 delG, 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. For the
amplification of mitochondrial DNA fragments, PCR method followed by pyrosequencing, with earlier
[9]
described primers and conditions were used . In brief, to quantitatively evaluate mutant allele, a method
of pyrosequencing was adapted for the condition when normal and mutant alleles may be present in a
biological specimen; the defective allele was quantified by analyzing the peak heights in the pyrogram of
one-chained PCR-fragments of a mitochondrial genome [9-11] .

Statistical analysis
Data processing was performed using the SPSS software package, version 22.0 (IBM Corp., Chicago, IL,
USA). Subprograms of descriptive statistics, variational analysis, parametric and nonparametric statistics,