Determination of selected oxysterol levels, oxidative stress, and macrophage activation indicators in children and adolescents with familial hypercholesterolemia

Abstract

Aim: Elevated levels of cholesterol in the bloodstream, also referred to as hypercholesterolemia, pose a significant risk for the onset of cardiovascular and cerebrovascular diseases. Oxysterols, cholesterol-derived oxidized compounds that form enzymatically or non-enzymatically, contribute to the development of atherosclerosis and coronary artery disease. This study aimed to examine the critical oxysterol levels in children and adolescents with hypercholesterolemia and explore the correlation between these levels, oxidative stress, and atherosclerosis progression.

Materials and methods: The study included 20 patients with familial hypercholesterolemia (FH) and 20 healthy individuals aged between 6 and 18 years. Participants were categorized into children (6-9 years) and adolescents (10-18 years). Pediatric and adolescent patients were selected from among subjects with LDL-C ≥ 130 mg/dL and diagnosed with heterozygous familial hypercholesterolemia (HeFH) based on the presence of mutations in the LDL receptor (LDL-R) gene. Patients with HeFH who were receiving regular atorvastatin therapy were included in the study.

Results: There were no notable differences in catalase and paraoxonase (PON1) activities among the groups. However, the patient group displayed substantially higher levels of malondialdehyde (MDA) (P = 0.0108) and superoxide dismutase (SOD) activity (P = 0.0103). Compared to the healthy control group, serum chitotriosidase (CHITO) activity (P = 0.037) and chitinase 3-like protein 1 (YKL-40) levels (P = 0.0027) were significantly elevated in the patient group. Furthermore, the carotid intima-media thickness (CIMT) measurements of the patient group were significantly greater than those of the healthy group (**P < 0.0001****). The patient group exhibited significantly elevated levels of 5,6-α-epoxycholesterol, Cholestane-3β,5α,6β-triol (C-triol), and 7-ketocholesterol (7-KC), whereas 27-hydroxycholesterol (27-OHC) was significantly more abundant in the healthy group. On the other hand, while 27-OHC/Total cholesterol (Total-C) levels were significantly higher in healthy individuals, the C-Triol/Total-C ratio was significantly higher in patients. No significant differences were found between the groups in terms of 7-KC/Total-C and 5,6-α-epoxycholesterol/Total-C levels.

Conclusion: This study highlights the key roles of oxysterols, oxidative stress, and macrophage activation in the development of atherosclerosis in pediatric and adolescent patients with FH. Elevated C-Triol levels in FH patients, alongside increased CIMT, point to early vascular changes despite atorvastatin therapy. In contrast, higher 27-OHC levels in healthy controls suggest differential oxysterol regulation due to cholesterol-lowering treatments in FH patients. C-Triol and 27-OHC/Total-C ratios showed potential as biomarkers to distinguish patients with FH. These findings emphasize the need for therapies targeting oxidative stress and macrophage activation in addition to cholesterol-lowering interventions.

Keywords: Atherosclerosis; CIMT; Hypercholesterolemia; Macrophage activation indicators; Oxidative stress; Oxysterol.

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

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

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

Fig. 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