Cardiovascular disease (CVD), including coronary artery disease (CAD) and ischemic stroke, is the leading cause of mortality worldwide (1). Multiple risk factors accelerate the development of CVD, including age, dyslipidemia, hypertension, smoking, hyperglycemia, and inflammatory factors (2-6). However, overall predictive value of the conventional cardiovascular risk factors is limited, and therefore there is an increasing need to identify novel risk factors for senility CVD (5, 6).
Several previous studies on amino acids have especially focused on their associations with obesity (7) and type 2 diabetes (T2D), a major risk factor for CVD (8-10). Previously published studies on the association of amino acids with CAD, ischemic stroke, and CVD (11-19) have been heterogeneous with respect to size of the study (small, large), study design (cross-sectional case-control studies, prospective studies), endpoints (CAD, ischemic stroke, or combination of both).
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T2D, CAD, and ischemic stroke share several risk factors, but the role of amino acids remains unclear in the development of cardiovascular events. Previous prospective studies have not systematically compared the significance of amino acids as predictors of CAD, stroke, and CVD in a large prospective population-based study. Therefore, the aim of our study was to investigate the association of serum concentrations of 9 amino acids (alanine, glutamine, glycine, histidine, isoleucine, leucine, phenylalanine, tyrosine, and valine), with the risk CAD, ischemic stroke, and CVD in a large longitudinal population-based Metabolic Syndrome in Men (METSIM) study (20).
Study Population
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The METSIM study includes 10 197 men, aged from 45 to 73 years, and randomly selected from the population register of Kuopio, Eastern Finland. At baseline, each participant had an outpatient visit to the Clinical Research Unit at the University of Kuopio. The design and methods of the METSIM study have been previously described in detail (21).
A total of 9584 men including 1412 men who had T2D were included in the current study. Participants with type 1 diabetes (n = 25) were excluded from current analyses. Altogether, 602 CVD events before the baseline study were recorded, including 404 CAD and 198 ischemic stroke events. These participants were excluded from all statistical analyses. We measured height and weight to the nearest 0.5 cm and 0.1 kg, respectively. Body mass index (BMI) was calculated as weight (kilograms) divided by height (meters) squared. We measured waist circumference at the midpoint between the lateral iliac crest and lowest rib. Smoking status was defined as current smoking (yes vs no). Diagnosis of hypertension was based on the use of antihypertensive medication. All participants underwent a 2-hour oral glucose tolerance test (75 g of glucose), and samples for plasma glucose and insulin were drawn at 0, 30, and 120 minutes. Diabetes was defined as fasting plasma glucose ≥7.0 mmol/L and/or 2-hour plasma glucose ≥11.1 mmol/L, and/or hemoglobin A1c ≥6.5%) and/or medication for diabetes according to the American Diabetes Association 2003 criteria (22).
Laboratory Measurements
We measured plasma glucose, insulin, total triglycerides, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and serum high-sensitivity C-reactive protein (hs-CRP), as previously described (23). We measured serum metabolites and lipoprotein lipids using a high-throughput serum nuclear magnetic resonance (NMR) platform operating at 500 MHz. Fasting serum samples collected at the baseline study were stored at -80°C and thawed overnight in a refrigerator before sample preparation. Aliquots of each sample (300 μL) were mixed with sodium phosphate buffer (300 μL). Details of the NMR experimentation have been described previously (24, 25).
Diagnosis of CAD and Ischemic Stroke Events
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We identified nonfatal and fatal CAD and ischemic stroke events from the hospital discharge registry of Kuopio University Hospital, which is the only hospital and cardiology outpatient clinic treating patients in the Kuopio region. Thereafter, 2 cardiologists (R.J., J.K.) reviewed medical records of all subjects with diagnosis of CAD based on the codes I22-I24 in the International Classification of Diseases according to internationally accepted criteria (26). Causes of CAD death were confirmed from death certificates obtained from the National Causes-of-Death Register of Statistics Finland. We identified 662 nonfatal or fatal CAD events, 394 nonfatal or fatal ischemic stroke events based on the codes I63-I69 in the International Classification of Diseases, and 966 nonfatal or fatal CVD events (CAD and stroke events combined). Incident cases of CVD events were recorded from the baseline study visit until February 28, 2021, with a mean follow-up period of 12.3 years.
Statistical Analyses
We performed statistical analyses using SPSS version 27 (SPSS, Chicago, IL). We log-transformed all continuous variables for statistical analyses, except for age and LDL-C. We used independent samples t test and χ 2 test to assess statistical significance between the 2 groups. We applied Cox regression analysis to evaluate baseline concentrations of the nine amino acids as predictors for CAD, ischemic stroke, and CVD events. Results of Cox regression analyses are presented without and with adjustment for confounding factors (age, diabetes status, fasting insulin, smoking, BMI, LDL-C, HDL-C, statin treatment, drug treatment for hypertension, and hs-CRP). Hazard ratio (HR) values are shown as per 1 SD increase in amino acid concentrations. The association with outcomes could be reasonably fit by a linear model. We applied a C-index (27) that is a standard measure of the predictive accuracy of a logistic regression model. To calculate C-index we first included confounding factors listed above in the Cox model (model 1) and then we added in the Cox model 3 amino acids (phenylalanine, tyrosine, alanine) (model 2). Correlations were calculated by Pearson correlations. Bonferroni-corrected limit for statistically significance was P < 0.0056 given 9 amino acids included in statistical analyses. P < 0.05 was considered as nominally significant.
Ethics Statement
The study protocol was approved by the Ethics Committee of University of Eastern Finland and Kuopio University Hospital. The study was conducted in accordance with the Helsinki Declaration. All study participants gave written informed consent.
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