Metabolomics profile and 10-year atherosclerotic cardiovascular disease (ASCVD) risk score.

Background: The intermediate metabolites associated with the development of atherosclerotic cardiovascular disease (ASCVD) remain largely unknown. Thus, we conducted a large panel of metabolomics profiling to identify the new candidate metabolites that were associated with 10-year ASCVD risk. Methods: Thirty acylcarnitines and twenty amino acids were measured in the fasting plasma of 1,102 randomly selected individuals using a targeted FIA-MS/MS approach. The 10-year ASCVD risk score was calculated based on 2013 ACC/AHA guidelines. Accordingly, the subjects were stratified into four groups: low-risk (n = 620), borderline-risk (n = 110), intermediate-risk (n = 225), and high-risk (n = 147). 10 factors comprising collinear metabolites were extracted from principal component analysis. Results: C4DC, C8:1, C16OH, citrulline, histidine, alanine, threonine, glycine, glutamine, tryptophan, phenylalanine, glutamic acid, arginine, and aspartic acid were significantly associated with the 10-year ASCVD risk score (p-values ≤ 0.044). The high-risk group had higher odds of factor 1 (12 long-chain acylcarnitines, OR = 1.103), factor 2 (5 medium-chain acylcarnitines, OR = 1.063), factor 3 (methionine, leucine, valine, tryptophan, tyrosine, phenylalanine, OR = 1.074), factor 5 (6 short-chain acylcarnitines, OR = 1.205), factor 6 (5 short-chain acylcarnitines, OR = 1.229), factor 7 (alanine, proline, OR = 1.343), factor 8 (C18:2OH, glutamic acid, aspartic acid, OR = 1.188), and factor 10 (ornithine, citrulline, OR = 1.570) compared to the low-risk ones; the odds of factor 9 (glycine, serine, threonine, OR = 0.741), however, were lower in the high-risk group. "D-glutamine and D-glutamate metabolism", "phenylalanine, tyrosine, and tryptophan biosynthesis", and "valine, leucine, and isoleucine biosynthesis" were metabolic pathways having the highest association with borderline/intermediate/high ASCVD events, respectively. Conclusions: Abundant metabolites were found to be associated with ASCVD events in this study. Utilization of this metabolic panel could be a promising strategy for early detection and prevention of ASCVD events.

The association between acylcarnitine and amino acids profile and metabolic syndrome and its components in Iranian adults: Data from STEPs 2016.

Background: Evidence, albeit with conflicting results, has suggested that cardiometabolic risk factors, including obesity, type 2 diabetes (T2D), dyslipidemia, and hypertension, are highly associated with changes in metabolic signature, especially plasma amino acids and acylcarnitines levels. Here, we aimed to evaluate the association of circulating levels of amino acids and acylcarnitines with metabolic syndrome (MetS) and its components in Iranian adults. Methods: This cross-sectional study was performed on 1192 participants from the large-scale cross-sectional study of Surveillance of Risk Factors of non-communicable diseases (NCDs) in Iran (STEP 2016). The circulating levels of amino acids and acylcarnitines were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in individuals with MetS (n=529) and without MetS (n=663). Results: The higher plasma levels of branched-chain amino acids (Val, Leu), aromatic amino acids (Phe, Tyr), Pro, Ala, Glu, and the ratio of Asp to Asn were significantly associated with MetS, whereas lower circulating levels of Gly, Ser, His, Asn, and citrulline were significantly associated with MetS. As for plasma levels of free carnitine and acylcarnitines, higher levels of short-chain acylcarnitines (C2, C3, C4DC), free carnitine (C0), and long-chain acylcarnitines (C16, C18OH) were significantly associated with MetS. Principal component analysis (PCA) showed that factor 3 (Tyr, Leu, Val, Met, Trp, Phe, Thr) [OR:1.165, 95% CI: 1.121-1.210, P<0.001], factor 7 (C0, C3, C4) [OR:1.257, 95% CI: 1.150-1.374, P<0.001], factor 8 (Gly, Ser) [OR:0.718, 95% CI: 0.651-0.793, P< 0.001], factor 9 (Ala, Pro, C4DC) [OR:1.883, 95% CI: 1.669-2.124, P<0.001], factor 10 (Glu, Asp, C18:2OH) [OR:1.132, 95% CI: 1.032-1.242, P= 0.009], factor 11 (citrulline, ornithine) [OR:0.862, 95% CI: 0.778-0.955, P= 0.004] and 13 (C18OH, C18:1 OH) [OR: 1.242, 95% CI: 1.042-1.480, P= 0.016] were independently correlated with metabolic syndrome. Conclusion: Change in amino acid, and acylcarnitines profiles were seen in patients with MetS. Moreover, the alteration in the circulating levels of amino acids and acylcarnitines is along with an increase in MetS component number. It also seems that amino acid and acylcarnitines profiles can provide valuable information on evaluating and monitoring MetS risk. However, further studies are needed to establish this concept.

Association of plasma acylcarnitines and amino acids with hypertension: A nationwide metabolomics study.

BACKGROUND: Identification of metabolomics profile in subjects with different blood pressure, including normal blood pressure, elevated blood pressure, stage 1 hypertension, and stage 2 hypertension, would be a promising strategy to understand the pathogenesis of hypertension. Thus, we conducted this study to investigate the association of plasma acylcarnitines and amino acids with hypertension in a large Iranian population. METHODS: 1200 randomly selected subjects from the national survey on the Surveillance of Risk Factors of Non-Communicable Diseases in Iran (STEPs 2016) were divided into four groups based on the ACC/AHA hypertension criteria: normal blood pressure (n = 293), elevated blood pressure (n = 135), stage 1 hypertension (n = 325), and stage 2 hypertension (n = 447). Plasma concentrations of 30 acylcarnitines and 20 amino acids were measured using a targeted approach with flow-injection tandem mass spectrometry. Univariate and multivariate logistic regression analysis was applied to estimate the association between metabolites level and the risk of hypertension. Age, sex, BMI, total cholesterol, triglyceride, HDL cholesterol, fasting plasma glucose, use of oral glucose-lowering drugs, statins, and antihypertensive drugs were adjusted in regression analysis. RESULTS: Of 50 metabolites, 34 were associated with an increased likelihood of stage 2 hypertension and 5 with a decreased likelihood of stage 2 hypertension. After full adjustment for potential confounders, 5 metabolites were still significant risk markers for stage 2 hypertension including C0 (OR = 0.75; 95%CI: 0.63, 0.90), C12 (OR = 1.18; 95%CI: 1.00, 1.40), C14:1 (OR = 1.20; 95%CI: 1.01, 1.42), C14:2 (OR = 1.19; 95%CI: 1.01, 1.41), and glycine (OR = 0.81; 95%CI: 0.68, 0.96). An index that included glycine and serine also showed significant predictive value for stage 2 hypertension after full adjustment (OR = 0.86; 95%CI: 0.75, 0.98). CONCLUSIONS: Five metabolites were identified as potentially valuable predictors of stage 2 hypertension.

Evaluating the association between amino acid and acylcarnitine profiles and different levels of coronary artery disease risk in postmenopausal women using targeted metabolomics technique.

OBJECTIVES: Postmenopausal women are at increased risk of developing coronary artery disease (CAD). Metabolomic approaches aim at discovering more helpful biomarkers of CAD to reduce the disease burden in the future. Here, we intend to find potential blood biomarkers, amino acids, and acylcarnitines in postmenopausal women with different severity of CAD by using high-throughput methods. METHOD: This cross-sectional study was performed on postmenopausal women ( n = 183) who underwent coronary CT scans. Coronary artery calcium scoring (CACS) was assessed to detect plaque burden and degree of coronary artery obstruction. The participants were divided into three groups based on the score as follows (i) "low CACS" ( n = 96); a score of 0 to 10, (ii) "medium CACS" ( n = 35); a score between 11 and 100 and (iii) "high CACS" ( n = 52); a score greater than 100. Metabolites, including amino acids and acylcarnitines, were quantified using a targeted mass spectrometry method in serum samples. The association between metabolites and disease status was evaluated using univariate and multivariate regression analyses with adjustment for confounding factors. Factor analysis was used to deal with multiple comparisons. RESULTS: Metabolites, including proline, glutamic acid, and phenylalanine, were significantly lower in the high CACS group than the low CACS one. Also, a lower level of lysine and phenylalanine in high CACS compared with medium one was observed. Concerning acylcarnitines, it was found that C4 and C8:1 significantly were higher in women with high CACS. The logistic regression analysis revealed that the circulating levels of these metabolites (except C4) were associated with the presence of coronary artery calcification independently of age, body mass index, and time of menopause. Also, the amino acids were associated independently of medication and diabetes. CONCLUSIONS: The present study indicated that circulating levels of amino acids and acylcarnitines profile in postmenopausal women are partly associated with the severity of CAD in these participants.

Metabolic signatures of insulin resistance in non-diabetic individuals.

BACKGROUND: Insulin resistance (IR) evolved from excessive energy intake and poor energy expenditure, affecting the patient's quality of life. Amino acid and acylcarnitine metabolomic profiles have identified consistent patterns associated with metabolic disease and insulin sensitivity. Here, we have measured a wide array of metabolites (30 acylcarnitines and 20 amino acids) with the MS/MS and investigated the association of metabolic profile with insulin resistance. METHODS: The study population (n = 403) was randomly chosen from non-diabetic participants of the Surveillance of Risk Factors of NCDs in Iran Study (STEPS 2016). STEPS 2016 is a population-based cross-sectional study conducted periodically on adults aged 18-75 years in 30 provinces of Iran. Participants were divided into two groups according to the optimal cut-off point determined by the Youden index of HOMA-IR for the diagnosis of metabolic syndrome. Associations were investigated using regression models adjusted for age, sex, and body mass index (BMI). RESULTS: People with high IR were significantly younger, and had higher education level, BMI, waist circumference, FPG, HbA1c, ALT, triglyceride, cholesterol, non-HDL cholesterol, uric acid, and a lower HDL-C level. We observed a strong positive association of serum BCAA (valine and leucine), AAA (tyrosine, tryptophan, and phenylalanine), alanine, and C0 (free carnitine) with IR (HOMA-IR); while C18:1 (oleoyl L-carnitine) was inversely correlated with IR. CONCLUSIONS: In the present study, we identified specific metabolites linked to HOMA-IR that improved IR prediction. In summary, our study adds more evidence that a particular metabolomic profile perturbation is associated with metabolic disease and reemphasizes the significance of understanding the biochemistry and physiology which lead to these associations.

Circulating amino acids and acylcarnitines correlated with different CAC score ranges in diabetic postmenopausal women using LC-MS/MS based metabolomics approach.

BACKGROUND: Diabetes mellitus (DM) and its cardiovascular disease (CVD) complication are among the most frequent causes of death worldwide. However, the metabolites linking up diabetes and CVD are less understood. In this study, we aimed to evaluate serum acylcarnitines and amino acids in postmenopausal women suffering from diabetes with different severity of CVD and compared them with healthy controls. METHODS: Through a cross-sectional study, samples were collected from postmenopausal women without diabetes and CVD as controls (n = 20), patients with diabetes and without CVD (n = 16), diabetes with low risk of CVD (n = 11), and diabetes with a high risk of CVD (n = 21) referred for CT angiography for any reason. Metabolites were detected by a targeted approach using LC-MS/MS and metabolic -alterations were assessed by applying multivariate statistical analysis. The diagnostic ability of discovered metabolites based on multivariate statistical analysis was evaluated by ROC curve analysis. RESULTS: The study included women aged from 50-80 years with 5-30 years of menopause. The relative concentration of C14:1, C14:2, C16:1, C18:1, and C18:2OH acylcarnitines decreased and C18 acylcarnitine and serine increased in diabetic patients compared to control. Besides, C16:1 and C18:2OH acylcarnitines increased in high-risk CVD diabetic patients compared to no CVD risk diabetic patients. CONCLUSION: Dysregulation of serum acylcarnitines and amino acids profile correlated with different CAC score ranges in diabetic postmenopausal women. (Ethic approval No: IR.TUMS.EMRI.REC.1399.062).

Targeted metabolomics analysis of amino acids and acylcarnitines as risk markers for diabetes by LC-MS/MS technique.

Diabetes is a common chronic disease affecting millions of people worldwide. It underlies various complications and imposes many costs on individuals and society. Discovering early diagnostic biomarkers takes excellent insight into preventive plans and the best use of interventions. Therefore, in the present study, we aimed to evaluate the association between the level of amino acids and acylcarnitines and diabetes to develop diabetes predictive models. Using the targeted LC-MS/MS technique, we analyzed fasting plasma samples of 206 cases and 206 controls that were matched by age, sex, and BMI. The association between metabolites and diabetes was evaluated using univariate and multivariate regression analysis with adjustment for systolic and diastolic blood pressure and lipid profile. To deal with multiple comparisons, factor analysis was used. Participants' average age and BMI were 61.6 years, 28.9 kg/m2, and 55% were female. After adjustment, Factor 3 (tyrosine, valine, leucine, methionine, tryptophan, phenylalanine), 5 (C3DC, C5, C5OH, C5:1), 6 (C14OH, C16OH, C18OH, C18:1OH), 8 (C2, C4OH, C8:1), 10 (alanine, proline) and 11 (glutamic acid, C18:2OH) were positively associated with diabetes. Inline, factor 9 (C4DC, serine, glycine, threonine) and 12 (citrulline, ornithine) showed a reverse trend. Some amino acids and acylcarnitines were found as potential risk markers for diabetes incidents that reflected the disturbances in the several metabolic pathways among the diabetic population and could be targeted to prevent, diagnose, and treat diabetes.

Mass spectrometry with derivatization method for concurrent measurement of amino acids and acylcarnitines in plasma of diabetic type 2 patients with diabetic nephropathy.

BACKGROUND: Amino acids (AAs) and acylcarnitines play a key role in metabolic disease and can be used as biomarkers of various diseases such as malignancies, type 2 diabetes (T2D), insulin resistance, and cardiovascular diseases, therefore, designing an accurate and simple laboratory method that simultaneously measure both groups of substances, could improve the process of analytes quantification. In this research, a flow injection tandem mass spectrometry (FI-MS/MS) method for simultaneous measurement of AAs and acylcarnitines in addition to results of validation is explained. METHODS: Samples were mixed with internal standards and after derivatization (with butanolic-HCL), AAs, and acylcarnitines were quantified by tandem mass spectrometry (SCIEX API 3200). Analytical performance studies were designed based on the Clinical and Laboratory Standards Institute (CLSI) guidelines including precision, accuracy, linearity, and limit of detection-quantification (LOD-LOQ) experiments. Samples from patients with T2D in different stages of kidney disease were also analyzed to ensure the clinical usage of the method. RESULTS: Performance evaluation of the method demonstrated adequate results. The mean of estimated inter-assay precision (reported as a coefficient variation) for AAs and acylcarnitines were less than 8.7% and 12.3%, the estimated mean bias was below 8.8% and 10.2% respectively. LOD of analytes ranged between 0.6-10 µmol per liter (µmol/L) for AAs and 0.02-1 µmol/L for acylcarnitines. LOQ analytes showed a range of 2-25 µmol/L and 0.05-5 µmol/L for AAs and carnitine/acylcarnitines respectively. In diabetic patients sample analysis, a significant increase in acylcarnitines (C2, C4, C5DC, C6, C8, C10, C14) and citrulline with a significant decrease in valine were seen in patients with severely increased albuminuria. CONCLUSION: FI-MS/MS method with pre-injection derivatization with butanolic-HCL can be used for concurrent measurement of AAs and carnitine/acylcarnitines in a short time and it satisfies the analytical performance requirements. This method is applied for AAs and carnitine/acylcarnitines measurement in patient with T2DM and results show some of the acylcarnitines and AAs can be involved in diabetic nephropathy development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40200-021-00786-3.