Demographic data and serum parameters

The age of the children and adolescents included in the study ranged from 6 to 18 years. Twenty patients who were diagnosed with heterozygous HeFH, as confirmed by the presence of mutations in the LDL receptor (LDL-R) gene and were receiving regular atorvastatin therapy, and 20 healthy controls were included in the study. The demographic data and serum parameters of the control and patient groups included in the study are presented in Table 1. There was no significant difference between the groups in the sex, body mass index, and age of the children and adolescents. When routine biochemical parameters in the serum were examined, total cholesterol (P < 0.0001) and LDL-C (P < 0.0001) levels were significantly higher in the patient group than in the control group, whereas HDL-C (P = 0.0153) levels were significantly higher in the control group than in the patient group. Although serum creatinine levels were similar, they were significantly higher in the control group than in the patient group (P = 0.037). There were no significant differences in other biochemical parameters between the groups.

Table 1 Demographic data and serum biochemical parameters of the study groups. Except for the serum cholesterol and creatinine levels, there were no significant differences between the two groupsOxidant stress and antioxidant capacity parameters

Table 2 presents findings related to MDA as an oxidative stress indicator and SOD, catalase, and paraoxonase activities as parameters of antioxidant capacity. There was no significant difference in catalase and PON1 activities between the groups. The MDA levels (P = 0.0108) and SOD activity (P = 0.0103) were significantly higher in the patient group.

Table 2 The MDA, SOD, Catalase, and PON1 levels. There was a significant difference only in the MDA and SOD levels between the groupsMacrophage activation indicators and carotid intima media thickness findings

The serum chitotriosidase activity and YKL-40 levels were examined as indicators of macrophage activation. The results are shown in Fig. 1A and B. Compared with the healthy control group, the patient group had significantly higher serum chitotriosidase activity (P = 0.037) and significantly higher YKL-40 levels (P = 0.0027).

The CIMT was determined by a cardiologist through echocardiographic evaluation to investigate the effect of atherosclerosis. The CIMT values for the patient and control groups are shown in Fig. 1C. As shown in Fig. 1C, the CIMT of the patient group was approximately 1.5 times that of the healthy group, and this difference was statistically significant (P < 0.0001).

Fig. 1

Comparison of Serum CHITO Activity, YKL-40 and CIMT levels. The CHITO activity, YKL-40 levels, and CIMT values were significantly higher in the FH group than in the healthy group. FH: Familial hypercholesterolemia, HC: Healthy Control

Plasma oxysterol levels

The DMAB derivatization method developed by Boenzi et al. was used to determine the plasma oxysterols. Figure 2 shows representative chromatograms of these oxysterols. Although 5,6-α-epoxychol and 27-OHC had the same multiple reaction monitoring (MRM) values, they were easily determined owing to their different retention times during chromatographic separation. The partial validation parameters for the method, including precision, accuracy, and linear detection range, are listed in Table S2.

Fig. 2

Representative chromatograms of DMAB derivatives of C-triol, 7-KC, 5,6-epoxychol, and 27-OHC (100 ng/mL each). Extract ion: C-triol m/z 534.3/132.1 and C-triol D7 m/z 541.3/132.1 at retention time of 1.48 min; 7-KC m/z 514.3/132.1 and 7-KC D7 m/z 521.3/132.1 at retention time of 1.48 min; 27-OHC m/z 516.3/132.1 at retention time of 1.24 min and 5,6-epoxychol m/z 516.3/132.1 at retention time of 1.69 min

Table 3 shows a comparison of plasma oxysterol levels between the hypercholesterolemic and healthy control groups. Except for 27-OHC, oxysterols were significantly higher in the patient group. However, 27-OHC levels were significantly higher in the healthy group (P = 0.0042). The values obtained by dividing the levels of oxysterols by the total cholesterol showed some differences compared with the values obtained without division. The 5,6-α-epoxychol was found to be significantly higher in the patient group (P = 0.0002****); however, when the ratio of 5,6-α-epoxychol to Total-C was compared, no difference was found between the groups (P = 0.2766). In the case of 27-OHC, there were significantly higher levels in favor of healthy individuals, whereas the significance level of 27-OHC/Total-C was greatly increased (P < 0.0001****). Both C-Triol and C-Triol/Total-C levels were found to be extremely significantly higher in the patient group (P < 0.0001****). However, when 7-KC was compared to total cholesterol in the patient group, this significant difference disappeared.

Table 3 Plasma oxysterol levels in both groups. Significant correlations are indicated in red and bold font, and * indicates their degree of significance. According to the data, there was a statistically significant difference between the groups for all parameters, except 7-KC/Total-C and 5.6-α-epoxychol/Total-CCorrelation analysis of oxidative stress markers, oxysterols, and atherosclerosis indicators in pediatric and adolescent hypercholesterolemia

In this study, the findings showing the combined correlation of the studied parameters with age and CIMT values are presented in heatmaps in Figs. 3 and 4. While Fig. 3 shows the correlation analysis related to the FH group, Fig. 4 presents the correlation analysis for the HC group.

In the FH group, significant correlations were found between the CIMT and some variables. A positive relationship was identified between the CIMT and age (r = 0.53, P = 0.017). Additionally, significant positive correlations were observed between the CIMT and 27-OHC (r = 0.55, P = 0.012) and the 27-OHC/Total-C ratio (r = 0.50, P = 0.025). In the healthy group, however, only a positive significant relationship between CIMT and LDL-C levels was detected (r = 0.47, P = 0.036).

In the FH group, there was a strong positive correlation between Total-C and LDL cholesterol (r = 0.97, P < 0.001). Additionally, a significant positive correlation was observed between Total-C and 5,6-epoxycholesterol (r = 0.58, P = 0.008). In the HC group, a negative relationship between Total-C and age was observed (r = -0.68, P < 0.001). In this group, a strong positive correlation was found between Total-C and LDL cholesterol (r = 0.90, P < 0.001), and positive correlations were also identified between Total-C and catalase (r = 0.54, P = 0.013) as well as C-triol (r = 0.58, P = 0.007).

In the FH group, a significant positive correlation was detected between LDL cholesterol (LDL-C) and 5,6-epoxycholesterol (r = 0.54, P = 0.015). In the HC group, a strong negative relationship was observed between LDL-C and age (r = -0.70, P < 0.001). Significant positive relationships were found between LDL-C and both CIMT (r = 0.47, P = 0.036) and C-triol (r = 0.50, P = 0.024).

No significant correlations were found between HDL-C and any other variables in the FH group. In the HC group, the only variable significantly correlated with HDL-C was catalase (r = 0.45, P = 0.049).

In the FH group, the only variable significantly correlated with triglyceride levels was the 5,6-epoxycholesterol/Total-C ratio, with a significant negative correlation between the two parameters (r = -0.47, P = 0.035). No significant correlations were found between triglycerides and any other variables in the HC group.

In the FH group, the only variable significantly correlated with SOD levels was catalase, with a strong negative correlation between the two enzymes (r = -0.84, P < 0.001) (Fig. 3). This suggests that SOD and catalase operate through different mechanisms related to oxidative stress. In the HC group, a significant negative correlation between SOD and catalase was also found (r = -0.49, P = 0.027).

In the FH group, the only variable significantly correlated with catalase levels was SOD, with a strong negative correlation between these two enzymes (r = -0.84, P < 0.001) (Fig. 3), indicating that oxidative stress is managed through different mechanisms. In the HC group, the catalase levels were significantly negatively correlated with the 7-ketocholesterol/Total-C ratio (r = -0.46, P = 0.040), which is distinct from the correlations observed in the FH group (Fig. 4).

No significant correlations were found between the CHITO levels and any variables in either the FH or HC group.

In the FH group, the only variable significantly correlated with the YKL-40 levels was the 27-OHC/Total-C ratio, with a significant negative correlation between these two parameters (r = -0.49, P = 0.030). This suggests that the YKL-40 levels may be related to oxysterol metabolism. No significant correlations were found between YKL-40 and any variables in the HC group.

No significant correlations were found between the PON-1 levels and any variables in the FH group. In the HC group, however, significant negative correlations were observed between the PON-1 levels and both C-triol (r = -0.49, P = 0.029) and the C-triol/Total-C ratio (r = -0.65, P = 0.002).

In the FH group, the variables significantly correlated with 5,6-epoxycholesterol levels were as follows: there was a positive correlation with Total-C (r = 0.58, P = 0.008), a significant positive relationship with LDL-C (r = 0.54, P = 0.015), and a positive correlation with 27-OHC (r = 0.55, P = 0.011). A strong positive correlation was also identified between 5,6-epoxycholesterol and the 5,6-epoxycholesterol/Total-C ratio (r = 0.69, P = 0.001).

In the HC group, the variables significantly correlated with 5,6-epoxycholesterol levels were as follows: a positive correlation with 27-OHC (r = 0.70, P < 0.001), a very strong positive relationship with the 5,6-epoxycholesterol/Total-C ratio (r = 0.91, P < 0.001), and a significant positive correlation with the 27-OHC/Total-C ratio (r = 0.46, P = 0.044).

Additionally, in the FH group, there was a strong positive correlation between 27-OHC and the 27-OHC/Total-C ratio (r = 0.82, P < 0.001). A significant negative correlation was found between 27-OHC and the C-triol/Total-C ratio (r = -0.55, P = 0.012). In the control group, a negative correlation was observed between the 27-OHC and age (r = -0.53, P = 0.016), while a strong positive correlation was identified between the 27-OHC and the 27-OHC/Total-C ratio (r = 0.81, P < 0.001).

Aside from these, in both groups, positive relationships were found between oxysterols and their ratios to Total-C.

Fig. 3

Heatmap showing significant correlations (P < 0.05) between variables in the familial hypercholesterolemia (FH) group. The heatmap visualizes significant correlations between parameters such as age, carotid intima-media thickness (CIMT), lipid profile (Total-C, LDL-C, HDL-C, Triglycerides), oxidative stress markers (MDA, SOD, Catalase), and oxysterols (5,6-epoxycholesterol, 27-OHC, C-Triol, 7-KC). Positive correlations are shown in shades of red, and negative correlations in shades of blue. The correlation coefficient (r) and significance level (P) are indicated in each cell

Fig. 4

Heatmap showing significant correlations (P < 0.05) between variables in the control (HC) group. In the HC group, the relationships between variables such as age, carotid intima-media thickness (CIMT), and lipid profile showed fewer significant correlations compared to the FH group. The negative correlation between the CIMT and HDL cholesterol is notable. The correlation coefficient (r) and significance level (P) are indicated in each cell

Diagnostic performance of CIMT and oxysterols: ROC curve analysisFig. 5

The ROC curve analysis for using individual markers between FH and control subjects. The ROC curve shows an area under the curve of 0.99 for CIMT, compared to 0.83 for 5,6-epoxychol, 0.74 for 27-OHC, 0.76 for 7-KC, 0.98 for C-Triol, 0.60 for 5,6-epoxy/total-c, 0.98 for 27-OHC-Total-C, 0.88 for C-Triol/total-c and 0.5 for 7-KC/total-C. shows. The highest sensitivities and specificities were obtained for CIMT, C-Triol, and 27-OHC/Total-C

Figure 5; Table 4 show the ROC curves comparing the individual markers. Upon examination of Fig. 5, it can be seen that the C-Triol and 27-OHC/ Total-C provide excellent discrimination equivalent to CIMT between the two groups. Additionally, the 5,6-α-epoxycholesterol and C-Triol/Total-C provided good discrimination between the two groups, whereas the discrimination power of 27-OHC and 7-KC was moderate. The 5,6-α-epoxychol/Total-C provides weak discrimination between the two groups, and there was no discrimination power of 7-KC/Total-C between the two groups. According to the findings presented in Table 4, the C-Triol appeared to be the most promising single biomarker in oxysterols to distinguish patients with FH, with an AUC of 0.985 (CI 95%; 0.944–1.00) and sensitivity and specificity values of 95% and 100%, respectively (P < 0.0001) (Fig. 4). In comparison, the CIMT exhibited an AUC of 0.991 (CI 95%; 0.971–1.00) and sensitivity and specificity of 95.6% and 93.33%, respectively (P < 0.0001) (Fig. 4).

Table 4 The area under the curve of oxysterols and CIMT when comparing the FH and HC groups