Introduction
Homocysteine (Hcy) is a sulfur-containing amino acid. The metabolism of Hcy is influenced by folic acid and vitamin B12, deficiencies of which can lead to high Hcy levels in the blood.1 ,2 In 1969, McCully3 made initial observations linking plasma Hcy concentrations and arteriosclerotic vascular disease. Many subsequent studies have shown that high Hcy levels may present a risk factor for atherosclerosis;4–6 however, there is little consensus among epidemiological investigations or case–control studies.6–12 These generally involved using participants from economically developed areas,6–9 where most participants demonstrated normal or slightly elevated Hcy levels.6–11 As a result, it remains unclear if high Hcy levels are related to atherosclerosis onset.
Lvliang city is one of the poorest areas of Shanxi Province, China. It has the highest incidence of neural tube defects (NTDs) in the world.13 NTDs may be related to local environmental conditions and eating habits.14 The use of folic acid has significantly reduced the incidence of NTDs in pregnant women in Lvliang city.14 Therefore, it was hypothesised that blood folic acid levels may be low while the levels of Hcy may be high among individuals aged 55 years and older. The aim of this cross-sectional study was to describe the relationship between carotid atherosclerosis, as assessed by ultrasonography, and serum concentrations of Hcy in this region.
Methods
This study was approved by the Research Ethics Committee of the Shanxi Medical University. Written informed consents were obtained from the patients.
Participants
From August to November 2012, a team of researchers (physicians and senior medical students) from the First Hospital of Shanxi Medical University travelled to Lvliang, Shanxi Province of China to study villagers born before 1 January 1958 who resided at least 2 months of the year in one of the four towns located there; namely, Xiajiaying, Kangchen, Gaojiagou or Caijiaya. A population of 9286 people aged 55 years and older was provided by the household registry department in each town. Of the 3005 participants who volunteered to complete the study questionnaires, 2304 completed carotid ultrasound examinations and blood tests. Thirteen participants were excluded because of incomplete data, and the remaining 2291 were evaluated, of which 1016 were men and 1275 were women.
A survey was carried out in the village clinics. Two days before the survey, villagers were informed of the survey through posters, radio advertising and telephone. For those volunteers who agreed to participate in the study, a face-to-face structured questionnaire was administered by the medical research team. After signing an informed consent form, participants underwent carotid ultrasound examinations and blood tests. Participants for statistical analysis included those who completed the questionnaire, carotid ultrasound examination and blood tests.
Data collection
The collected survey data included demographic information and cerebrovascular disease risk factors. Demographic characteristics included gender, date of birth, education, occupation and marital status. Cerebrovascular disease risk factors included history of smoking, hypertension and diabetes. Participants’ blood pressure (BP), height and weight were recorded. A qualified nurse took a fasting venous blood sample. Information regarding the participants’ medical history was self-reported or obtained through medical records. For the purposes of this study, history of smoking was defined as smoking continuously for 6 months or more and within 30 days prior to the survey.15 History of drinking was defined as a daily alcohol intake of more than 25 mg (80 mL of liquor, 200 mL of wine and 600 mL of beer).16
For each participant, the same researcher measured the BP twice, between 08:00 and 10:00, with the participant seated and using the same calibrated sphygmomanometer. Participants were asked to rest for at least 15 min, not to smoke and to empty their bladder up to 30 min beforehand. Average values of the systolic and diastolic BP (SBP and DBP, respectively) were used for analysis. Hypertension was defined as SBP≥140 mm Hg or DBP≥90 mm Hg, according to the 2010 Chinese guidelines for the management of hypertension. Height and weight were measured indoors. Body mass index (BMI; kg/m2) was calculated as an index of obesity.16 The WHO cut-off points of BMI<25.0 for non-obese and ≥25.0 for obese adults in Asian populations were used.
Biochemical determinations
Blood was drawn from the antecubital vein, to measure fasting blood glucose (FBG). Blood samples were centrifuged within 1 hour and frozen at −70°C. Blood samples were analysed at the First Hospital of Shanxi Medical University clinical laboratory. A Beckman UniCel DxC 800 Synchron Clinical System Analyzer (Beckman Coulter) was used to detect total cholesterol (TC), high-density lipoprotein (HDL) cholesterol (Immuno FS, DiaSys), triglycerides (TGs; Beckman Coulter) and uric acid (UA; Beckman Coulter). Low-density lipoprotein (LDL) cholesterol levels were calculated using the Friedewald equation for participants who had TG levels <400 mg/dL.
Hcy was measured using an enzyme cycling method with a Beckman UniCel DxC 800 Synchron Clinical System Analyzer (Beckman Coulter). All the participants were divided into four groups according to the Hcy level of four quantiles.
Measurement of carotid atherosclerosis
Carotid artery ultrasound scan was performed by a qualified sonographer, using a colour Doppler ultrasound scanner (Logic E, American GE). Participants were inspected in the supine position, with the head upright and the anterior portion of the neck fully exposed. The proximal internal carotid artery (ICA), ICA bulb and distal ICA were detected in sequence. The distance from the ICA lumen-intima border to the media-outer border was measured as the intima media thickness (IMT), in longitudinal images at the diastolic phase. The proximal part of the vessel wall to a plaque was measured, where there was a plaque. According to Mannheim carotid IMT consensus17 and carotid stenosis ultrasound diagnostic criteria,18 IMT≥1.0 mm was defined as IMT thickening. IMT≥1.5 mm or above the lumen was defined as a plaque. Participants showing one or several stenotic lesions, with a stenosis rate of <50% and no change in blood flow velocity (BFV), were not recorded. A stenosis rate of more than 50% and altered BFV was recorded. We defined IMT thickening and or carotid plaque(s) as carotid atherosclerosis.
Statistical analysis
Data were checked, verified and recorded using Epidata software (V.3.1). Results are reported as means±SDs. Quantitative variables between groups were compared by t-test, approximate t-test and variance analysis, whereas classifications of variable rate were compared using the χ2 test. Logistic regression was used to estimate ORs and 95% CIs. Statistical significance was defined as a p value <0.05. All statistical analyses were performed using SPSS (V.13.0).