Correlation between serum advanced glycation end products and vascular complications in type 2 diabetic patients

Vascular complications were correlated with AGE levels measured using serum from diabetic patients

The clinical characteristics of patients with diabetes and vascular complications (N = 154) are summarized in Table 1 and Supplementary Figure 1, Supplementary Tables 1 and 2. Serum samples from diabetic patients with and without macrovascular complications (non-Mac, N = 116; or Mac, N = 38), HbA1c, BMI, urinary albumin-creatinine ratio (ACR), triglycerides, low-density lipolysis. No differences were found in protein cholesterol (LDL-C), γ-glutamyl transpeptidase (GGT), or aspartate. aminotransferase (AST), systolic blood pressure (SBP), diastolic blood pressure (DBP), or plaque score. In addition, HbA1c, BMI, estimated glomerular filtration rate (eGFR), ACR, triglycerides, high-density lipoprotein cholesterol (HDL-C), GGT, AST, DBP, or plaque score also There was no difference. No microvascular complications (non-mic, N = 60; or mic, N = 94).

To assess the relevance of AGEs as indicators of macrovascular and microvascular complications in advanced diabetes, serum samples were processed with FSPES and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). were used to analyze free AGEs and total AGEs. Free among patients with macrovascular or microvascular complications (with: N = 103) or with nonvascular complications (without: N = 51) N^ε-(Carboxymethyl)lysine (CML), free N^ε-(Carboxyethyl)lysine (CEL), free N^δ-(5-Hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), total CML and total CEL levels were higher in patients with vascular complications than in patients with non-vascular complications (Wilcoxon rank sum test)) (Fig. 1c, d, e, h, i). However, free Lys, free Arg, total Lys, total Arg, and total MG-H1 levels remained unchanged (Fig. 1a, b, f, g, j). We analyzed the factors that influence the increase of AGEs in diabetic complications. eGFR as a confounder was correlated with variation in free CML (r = − 0.49, P< 0.001), free MG-H1 (r = − 0.5, P< 0.001), total CML (r = − 0.29, P= 0.0016) and total CEL (r = − 0.59, P< 0.001). Furthermore, free CML levels were correlated with free MG-H1 (r = 0.7, P< 0.001), and total CEL (r = 0.55, PFree MG-H1 levels were then correlated with total CEL (r = 0.55, P< 0.001), and total CML levels were correlated with total CEL (r = 0.72, P< 0.001), indicating a stronger positive correlation with the Spearman correlation (Figure 2). Each AGE showed positive correlations with other AGEs at different concentrations.

Correlation between increased AGEs and biochemical parameters in diabetic patients. Statistical analysis: Spearman analysis. The significance level was set at 0.05. Blue text indicates a positive correlation and red text indicates a negative correlation. FPG, fasting plasma glucose; BMI, body mass index. DM, duration of diabetes; ACR, urine albumin-creatinine ratio; TG, triglycerides; LDL, low-density lipoprotein cholesterol. HDL, high-density lipoprotein cholesterol. GGT, γ-glutamyl transpeptidase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; SBP, systolic blood pressure; DBP, diastolic blood pressure; Plaque, plaque score.

Free CML, free CEL, free MG-H1, total CML and total CEL were increased in patients presenting with macrovascular complications (Wilcoxon rank sum test) (Fig. 3c, d, e, h, i and Supplementary Table 3). However, free Lys, free Arg, total Lys, total Arg, and total MG-H1 levels remained unchanged (Fig. 3a, b, f, g, j).

Changes in AGE in macrovascular complications. (be) free squirrel, (b) free argument, (c) Free CML, (d) Free CEL, (e) Free MG-H1, (f) Total Lys, (g) total arguments, (h) Total CML, (I) Total CEL (j) Total MG-H1 levels were compared between non-Macs (N = 116) and Macs (N = 38). Data are displayed as bold horizontal bars in a boxplot indicating the median value. Statistical analysis: Wilcoxon rank sum test. *P< 0.05; **P< 0.01; ***P< 0.001.

In patients exhibiting microvascular complications, free CML, free CEL, free MG-H1, and total CML levels were increased compared to patients in the non-Mic group (Wilcoxon rank sum test) (Fig. 4c, d, e, h, and supplementary) Table 3). However, free Lys, free Arg, total Lys, total Arg, total CEL and total MG-H1 remained unchanged (Fig. 4a, b, f, g, i, j).

AGE variation in microvascular complications. (be) free squirrel, (b) free argument, (c) Free CML, (d) Free CEL, (e) Free MG-H1, (f) Total Lys, (g) total arguments, (h) Total CML, (I) Total CEL (j) Total MG-H1 levels were compared between non-Mic (N = 60) and Mic (N = 94). Data are displayed as bold horizontal bars in a boxplot indicating the median value. Statistical analysis: Wilcoxon rank sum test. *P< 0.05; **P< 0.01; ***P< 0.001.

Evaluation of serum AGE level by Z-score

AGEs were positively correlated with other AGEs at different concentrations, making it difficult to evaluate complications using a single AGE. Therefore, AGE z-scores were calculated by averaging and standardizing the increased AGE z-scores for each complication (Supplementary Table 3). For macrovascular complications, increased levels of free CML + free CEL +, and free MG-H1 were normalized to free AGE z-scores, whereas increased levels of total CML + and free CEL were associated with total AGE. Normalized to z-score. For microvascular complications, increased levels of free CML + , free CEL + , and free MG-H1 were normalized to free AGE z-score. However, the total AGE z-score was not calculated for microvascular complications because the increase in total AGE was limited to total CML. Free AGE and total AGE z-scores of patients exhibiting Mac were higher than non-Mac patients, and free AGE z-scores of patients exhibiting Mic were higher than non-Mic patients (Figure 5).

Variation in AGE Z score for each vascular complication. (be) AGE Z score between non-Mac and Mac (free), (b) AGE Z score (total) between non-Mac and Mac, (c) AGE z-score (free) between non-Mic and Mic is now listed. Data are displayed as bold horizontal bars in a boxplot indicating the median value. Statistical analysis: Wilcoxon rank sum test. *P< 0.05; **P< 0.01; ***P< 0.001.

We then analyzed the likelihood of AGE z-scores discriminating each complication using logistic regression. In patients presenting with macrovascular complications, Z-scores for both free and total AGE levels were significantly higher than Z-scores for single AGE levels in univariate logistic regression analysis (free CML: P= 0.107, free CEL: P= 0.144, free MG-H1: P= 0.039, total CML: P= 0.013, total CEL: P= 0.008, free AGE Z-score: P= 0.002, total AGE Z-score: P= 0.003) (Table 2a). However, in multivariate logistic regression analysis, there was no significant difference between free AGE z-score and total AGE z-score (free AGE z-score: P= 0.320, OR = 1.28, 95%CI = 0.79 to 2.09, total AGE Z-score: P= 0.267, OR = 1.31, 95%CI = 0.81–2.11, eGFR: P= 0.100, OR = 0.98, 95%CI = 0.96–1.00). In patients presenting with microvascular complications, the Z-score of free AGE was also more significant than single AGE in univariate logistic regression (free CML: P= 0.002, free CEL: P= 0.020, free MG-H1: P≤ 0.001, total CML: P= 0.044, free AGE Z-score: P≤ 0.001) (Table 2b). Additionally, free AGE z-scores showed significant differences in multivariable logistic regression (free AGE z-scores: P= 0.001, OR = 1.98, 95%CI = 1.32–3.09, eGFR: P= 0.883, OR = 1.00, 95%CI = 0.98–1.02). Finally, receiver operating characteristic (ROC) analysis was used to analyze the potential of AGE z-score as a diagnostic model for each complication. The area under the curve (AUC) for free AGE z-score was 0.70, whereas for total AGE z-score, the AUC was 0.65 for patients presenting with macrovascular complications (Fig. 6a). Meanwhile, for free AGE z-score, the AUC for patients exhibiting microvascular complications was 0.68 (Fig. 6b). In Mic patients, the AUC of AGE z-score was higher than that of single AGE (free CML: AUC = 0.66, free CEL: AUC = 0.62, free MG-H1: AUC = 0.67, total CML: AUC = 0.6). . . Additionally, the AUC score for HbA1c was lower than that for a single AGE level or AGE z-score for both complications (HbA1c for Mac: AUC = 0.57, HbA1c for Mic: AUC = 0.58). eGFR showed lower AUC scores than single AGE levels or AGE z-scores in patients exhibiting Mic (eGFR in Mac: AUC = 0.7, eGFR in Mic: AUC = 0.58).

ROC curves for AGE z-score and other parameters. (be) Free CML (Orange), Free CEL (Yellow), Free MG-H1 (Lime Green), Total CML (Green), Total CEL (Light Green), AGE z-Score (Free) (Light Blue), AGE z- ROC curve scores (total) (blue), HbA1c (purple), and eGFR (pink) for patients with macrovascular complications were created based on the values ​​of 154 cases. (b) Free CML (orange), free CEL (yellow), free MG-H1 (lime green), total CML (green), AGE Z-score (free) (light blue), HbA1c (purple), and eGFR (pink) microvessels The ROC curve values ​​for patients with complications were created based on the values ​​of 154 cases. AUC is displayed on the graph.