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

Lipids in Health and Disease volume 23, Article number: 374 (2024) Cite this article

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.

Introduction

Oxysterols are oxidized derivatives of cholesterol that play significant roles in various biological processes including cholesterol metabolism, inflammation, and cellular signaling [1, 2]. They are produced through enzymatic and non-enzymatic pathways, and can influence numerous physiological and pathological conditions, including cardiovascular diseases, neurodegenerative disorders, and metabolic syndromes [1, 3,4,5]. Cholestane-3β,5α,6β-triol (C-Triol), a polyoxygenated sterol, has garnered attention in recent research due to its significant role as a biomarker for Niemann-Pick disease type C (NPC) and its potential therapeutic applications. This compound is primarily derived from cholesterol through enzymatic processes involving cholesterol epoxide hydrolase, which converts 5,6-epoxycholesterol into C-Triol [6, 7]. The 7-KC is an oxidized derivative of cholesterol, classified as an oxysterol. It is formed through the oxidation of cholesterol, primarily at the C7 position, and is known for its significant biological activities and implications in various diseases, particularly cardiovascular diseases and atherosclerosis [8]. The 5,6α-epoxycholesterol (5,6-α-epoxychol) is a key autoxidation product of cholesterol [2, 7]. Research has indicated that 5,6-α-epoxycholesterol may serve as an important mediator in the development of atherosclerosis and related inflammatory diseases. It influences lipid metabolism and inflammatory responses, which are critical factors in the progression of atherosclerosis [9]. The 27-OHC is primarily produced through the enzymatic action of the cytochrome P450 enzyme CYP27A1, which hydroxylates cholesterol at the C27 position [1, 3]. This compound plays a significant role in various biological processes and has been implicated in several diseases, particularly cancer and cardiovascular diseases [9, 10].

Reactive Oxygen Species (ROS) are molecules containing oxygen that exhibit high reactivity. They are generated as byproducts of regular cellular metabolism, especially during mitochondrial respiration. While the ROS are crucial for cell signaling and maintaining homeostasis, their overproduction can cause oxidative stress, contributing to the development of various diseases such as cancer, cardiovascular conditions, and neurodegenerative disorders [11, 12]. Malondialdehyde (MDA) is a widely recognized indicator of lipid peroxidation, a process initiated when the ROS target polyunsaturated fatty acids within cell membranes, resulting in cellular damage [12, 13]. The MDA is formed as a by-product of this lipid peroxidation and is often used as a biomarker to assess oxidative stress in various clinical conditions [13]. Elevated levels of MDA have been associated with several pathological states, including chronic diseases such as diabetes, cardiovascular diseases, and neurodegenerative disorders [14, 15]. Superoxide Dismutase (SOD) is a crucial antioxidant enzyme that facilitates the conversion of superoxide radicals into hydrogen peroxide and oxygen, helping to reduce oxidative stress [16]. The SOD is vital for safeguarding cells against oxidative damage induced by the ROS. The SOD activity has been shown to correlate with the levels of oxidative stress markers such as the MDA in various studies, indicating its protective role against oxidative damage [17, 18].

Atherosclerosis is a complex inflammatory disease that involves various enzymes and structural changes that contribute to its development. Chitotriosidase (CHITO) is an enzyme associated with inflammation and immune responses, with elevated activity observed in individuals with atherosclerosis, indicating its potential as a marker for disease severity [19, 20]. On the other hand, paraoxonase 1 (PON1) is an enzyme linked to high-density lipoproteins (HDL) that plays a role in preventing the oxidation of low-density lipoproteins (LDL) and reducing the risk of atherosclerosis [21]. Recent studies have highlighted the importance of PON1 in limiting LDL cholesterol oxidation and in preventing atherosclerosis and stroke [22].

The CIMT is a critical indicator of atherosclerosis and reflects hypertrophy of the intima and media layers of arterial walls [23]. Recent research has emphasized the significance of CIMT as a marker for subclinical atherosclerosis and predictor of cardiovascular disease risk [24]. Studies have shown that an increased CIMT is associated with generalized atherosclerosis and can predict future cardiovascular events [19, 25]. Additionally, carotid plaques and focal thickening of the intima layer are strong predictors of cardiovascular disease [24]. The relationship between chitotriosidase activity, PON1 enzyme activity, and CIMT in the context of atherosclerosis development is intricate. Chitotriosidase activity is associated with the severity of atherosclerosis and macrophage activation [19, 26, 27]. On the other hand, PON1, along with HDL cholesterol, plays a crucial role in limiting LDL cholesterol oxidation and preventing atherosclerosis and stroke [22]. The CIMT serves as a structural marker that reflects arterial wall changes associated with atherosclerosis and is a reliable indicator of subclinical atherosclerosis and cardiovascular risk [24]. Recent studies have highlighted the interconnectedness of these factors to understand the pathophysiology of atherosclerosis and develop targeted interventions.

Hypercholesterolemia is a significant contributor to the development of atherosclerosis. Despite previous research demonstrating a connection between oxysterol levels and the progression of atherosclerosis, limited studies have focused on pediatric populations, particularly those with familial hypercholesterolemia (FH). This study hypothesizes that specific oxysterols, including 5,6-α-epoxycholesterol, 27-OHC, C-Triol, and 7-KC, play a direct role in oxidative stress and atherosclerosis progression in children with FH. Specifically, 5,6-α-epoxychol, formed through ROS interactions; 27-OHC, produced enzymatically; and C-Triol and 7-KC, which can be formed by both pathways, were measured using the LC-MS/MS method. These oxysterols were selected as they are among the nine most abundant in plasma and have been frequently studied for their role in atherosclerotic plaque formation [5]. By examining their association with oxidative stress markers and carotid intima-media thickness (CIMT), this study aims to elucidate which oxysterols contribute to early vascular changes in young patients with FH. The novel aspect of this research lies in its focus on a pediatric cohort, which has been underexplored in previous literature. This study uniquely contributes to the understanding of oxysterol regulation and its potential as a biomarker for early atherosclerosis detection in children with FH.

Materials and methods

Chemicals

C-triol, 5,6-α-epoxycholesterol (5,6-α-epoxychol), C-triol-d7, 27-OHC, 7-KC, and 7-KC-d7 were obtained from Avanti Polar Lipids (Alabaster, Alabama, USA). LC-MS grade ammonium formate, 1,1′-carbonyldiimidazole, 4-(dimethylamino)butyric acid (DMAB), water, methanol, acetonitrile, chloroform, dichloromethane, formic acid, and all remaining chemicals were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). YKL-40 Assay Kit was obtained from Bioscience (San Diego, California, USA).

Subjects/Patients

The study included 20 patients between the ages of 6–18 who were being followed up for familial hypercholesterolemia, receiving regular atorvastatin and diet therapy for managing their condition and 20 healthy individuals without any hyperlipidemia, all of whom were admitted to the Metabolism Department of the Pediatric Health and Diseases Department of Ege University Faculty of Medicine.

All pediatric patients included in the study were diagnosed with heterozygous familial hypercholesterolemia (HeFH), as confirmed by the presence of mutations in the LDL receptor (LDL-R) gene. Each patient exhibited low-density lipoprotein cholesterol (LDL-C) levels exceeding 130 mg/dL and was receiving regular atorvastatin therapy along with prescribed diet therapy for hypercholesterolemia. These patients were continuously treated and followed up at the Metabolism Unit of the Pediatric Health and Diseases Department, Ege University Faculty of Medicine. Both groups consisted of individuals with normal fasting glucose levels, no history of diabetes, cancer, renal failure, liver failure, and no chronic diseases other than hypercholesterolemia.

When patients and volunteers came to the clinic for routine check-ups or when a diagnosis was made for the first time (between the ages of 6–18), overnight fasting venous blood samples (3 mL of heparinized and 4 mL of plain blood samples) were collected. Samples were centrifuged at 3000 × g for 10 min to separate the serum, plasma, and hemolysates and stored at -80 °C until analysis. Intima-media thickness measurements of the patients were performed on the same day at the Pediatric Health and Diseases Department and Cardiology Department of Ege University Faculty of Medicine.

Demographic data of the patients (age, sex, weight, and clinical findings) were obtained during their clinic visit. The results of routine biochemical tests performed on the same day as blood samples taken from patients were obtained from clinical records.

Measurement of plasma oxysterol levels by LC-MS/MS method

The derivatization method developed by Boenzi et al. [28] was used to measure C-triol and 7-KC. The oxysterol standards were prepared by dissolving them in methanol at a concentration of 1 mg/mL and stored at -80 °C. Dilutions were performed using acetonitrile. The UPLC and MS-MS conditions of the method are provided in the supplementary file (Table S1).

Measurement of SOD

SOD activity was measured in erythrocyte hemolysates using the method developed by Sun et al. [29]. The principle of the method is as follows: xanthine oxidase generates superoxide radicals while oxidizing xanthine. The released superoxide radical reacts with nitroblue tetrazolium (NBT) to form a blue-colored formazan dye, which can be measured at 560 nm absorbance. SOD enzyme converts the generated superoxide to H2O2, preventing the formation of formazan dye. SOD activity was determined by measuring the decrease in absorbance. Details of the method, reagents used, and preparation of the hemolysate are provided in the supplementary file.

Measurement of catalase

Catalase activity in erythrocyte hemolysates was also measured using the method developed by Aebi [30]. The principle of the method is briefly as follows: H2O2 absorbs light at a wavelength of 240 nm. Catalase catalyzes the conversion of H2O2 to H2O and O2. Catalase activity was determined from the decrease in the absorbance of H2O2. Details of the method and reagents used are provided in the Supplementary File.

Measurement of serum MDA levels

MDA was measured in the serum using the method developed by Ohkawa et al. [31]. The principle of this method is based on measuring the absorbance of the pink-colored product formed as a result of the reaction between MDA and TBA at 532 nm [31]. The details of the method and the reagents used are provided in the supplementary file.

Determination of paraoxanase activity

The principle of the method is based on the catalysis of the hydrolysis of paraoxon (diethyl p-nitrophenyl phosphate) to p-nitrophenol and diethyl phosphate by paraoxonase present in the serum. The increase in absorbance of p-nitrophenol at 412 nm was measured spectrophotometrically [32]. Paraoxonase activity was calculated in U/mL using the molar extinction coefficient (18050). The details of the method and the reagents used are provided in the supplementary file.

Measurement of serum YKL-40 levels and CHITO activity

Serum YKL-40 levels were analyzed using the Human YKL-40 ELISA Kit (Lot number 202212, Bioscience, San Diego, California, USA) with a Thermo multiscan spectrofluorometer microplate reader, according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader.

Chito activity was determined using a fluorometric assay based on the method described by Hollak et al. [33]. In summary, 10 µL of serum was incubated with 4-methylumbelliferyl-β-D-NN, N’-triacetylchitotriose (Sigma Chemical, St. Louis, MO, USA) as the substrate in a 0.25 mol/L sodium acetate buffer (pH 5.5) at 37 °C for 1 h. The reaction was terminated by adding 0.1 mol/L ethylenediamine. The fluorescence of released 4-methylumbelliferone was measured using a fluorometer (Thermo Fisher Scientific/Specrofluorometer/Varioskan, MA, USA) with excitation at 365 nm and emission at 450 nm. A calibration curve was generated by using 4-methylumbelliferone. Enzymatic activity was expressed as nmol/mL/h. The coefficients of variation (CV) for within- and between-run precision were 8.8% and 16%, respectively.

Measurement of intima-media thickness

All ultrasonographic studies were performed by the same person, who was unaware of the laboratory results. Patients were first rested in a supine position for 10 min. High-resolution B-mode ultrasonography was performed in all patients using a 12 MHz linear probe (high resolution) from the right carotid artery, with the patient’s head slightly turned to the left, using an echocardiography device (GE Vingmed, Vivid 7.0, Ultrasound AS, Horten, Norway). Measurements were taken at end-diastole in the carotid artery of each patient, and the measurements for each patient were recorded. The average of the three measurements was used to determine the intima-media thickness.

Statistical analysis

Statistical analyses were conducted using GraphPad 8.3 and SPSS 24.0 software. A significance level of P < 0.05 was applied. Data with a normal distribution are presented as mean ± SD (with minimum and maximum values), while non-normally distributed data are shown as mean ± SD (with interquartile range). Normality was assessed using the Shapiro-Wilk test. Given the sample size of fewer than 30 participants, the Mann-Whitney U test was employed for comparisons between groups, and Spearman’s correlation test was used to analyze the relationship between variables, irrespective of the distribution. A P-value > 0.05 was considered not significant, P < 0.05 was deemed significant, P < 0.01 was considered highly significant, and P < 0.001 was considered extremely significant.

Results

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 groups
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Oxidant 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 groups
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Macrophage 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
figure 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

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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
figure 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

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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-C
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Correlation 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
figure 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

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Fig. 4
figure 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

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Diagnostic performance of CIMT and oxysterols: ROC curve analysis

Fig. 5
figure 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

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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
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Discussion

This study investigated the levels of specific oxysterols in children and adolescents with FH, focusing on their roles in oxidative stress, macrophage activation, and atherosclerosis progression. The findings reveal significant elevations in oxysterols, oxidative stress markers, and macrophage activation indicators in the FH group compared to the healthy controls, supporting the link between hypercholesterolemia, oxidative stress, and atherogenesis. The results also highlight significant differences in oxidative stress markers, macrophage activation indicators, and CIMT between the two groups, aligning with literature that emphasizes the role of cholesterol metabolism and its derivatives in atherosclerosis. The patient and control groups were distinguished by hypercholesterolemia, with Total-C (P < 0.0001) and LDL-C (P < 0.0001) levels showing clear and statistically significant differences.

The findings in Table 2 on MDA and antioxidant enzymes–SOD, catalase, and paraoxonase—highlight the oxidative stress and antioxidant defense mechanisms in the studied groups. Elevated MDA (P = 0.0108) and SOD activity (P = 0.0103) in the patient group reflect heightened oxidative stress, aligning with evidence that lipid peroxidation increases MDA levels [34]. The MDA is a known biomarker of oxidative stress, especially in cardiovascular diseases [35]. The negative correlation between SOD and catalase in patients with FH (r = -0.84, P < 0.001) suggests a compensatory mechanism—SOD reduces superoxide radicals to hydrogen peroxide, which catalase further detoxifies [36]. The strong negative correlation points to an imbalance in antioxidant defenses under high oxidative stress, potentially contributing to the pathological state of FH [37]. Similarly, a milder negative correlation in the control group (r = -0.49, P = 0.027) suggests these enzymes respond differently across varying oxidative environments [38]. Despite increased SOD activity in patients, elevated MDA levels suggest that the antioxidant response remains insufficient to fully neutralize oxidative stress, causing cellular damage [39]. This imbalance underscores the complexity of antioxidant defenses, where increased enzyme activity does not always translate to reduced oxidative damage. Such oxidative stress likely plays a role in the pathogenesis of cardiovascular complications in patients with FH, reinforcing the importance of effective antioxidant strategies [40].

The elevated serum CHITO activity and YKL-40 levels observed in the FH group indicate heightened macrophage activation, which is consistent with findings in other studies that have linked macrophage activation to atherosclerosis progression [41]. Chitotriosidase activity, which is an indicator of macrophage activation, has been shown to be altered in patients with FH. This is consistent with findings from previous studies that reported alterations in cholesterol metabolism within monocytes of these patients, highlighting the complex interplay between cholesterol homeostasis and immune cell function [42]. Macrophages play a pivotal role in the inflammatory response associated with atherosclerosis, and their activation is often exacerbated by elevated cholesterol levels [43]. As shown in Fig. 1, the YKL-40 level in the patient group is approximately 1.7 times higher than that of the healthy group (P = 0.00270). Although the difference in CHITO activity between the two groups is not as significant as that of YKL-40, it is approximately 1.3 times higher than that of the healthy group (P = 0.0370). As mentioned in the literature, increased macrophage activity is expected in the hypercholesterolemic group. In this study, no relationship was found between CHITO activity and blood lipids. Similarly, Canudas et al. compared the group using atorvastatin with the group using bezafibrate in patients with combined hyperlipidemia before and after treatment and found no significant correlation between CHITO and blood lipids [44]. In the study, the patient group was treated with atorvastatin. This result may suggest that statin drugs reduce macrophage activation in patients with hypercholesterolemia. No correlation was found between the YKL-40 levels and blood lipids. Kwon et al. also found no significant relationship between the YKL-40 and total cholesterol and LDL-cholesterol in their study of 479 children aged 10–12 years [45].

The 5,6-α-Epoxychol, a cholesterol oxidation product, has emerged as a key biomarker in understanding the atherogenic process, particularly in hypercholesterolemic conditions [46]. In this study, 5,6-α-epoxychol levels were significantly higher in the FH group compared to healthy controls, underscoring its role in the pathogenesis of atherosclerosis in FH. This finding aligns with prior studies reporting elevated oxysterols in hypercholesterolemia and their contribution to endothelial dysfunction and plaque formation [5, 47]. Although the ratio of 5,6-α-epoxychol to total cholesterol (Total-C) did not differ significantly between groups, this suggests that total cholesterol alone may not fully explain the oxidative changes seen in FH. Positive correlations between 5,6-α-epoxychol, Total-C, and LDL-C in the FH group further emphasize the involvement of cholesterol metabolism in oxysterol accumulation. Additionally, its correlation with 27-hydroxycholesterol (27-OHC)—another oxysterol implicated in atherosclerosis—highlights the interconnected nature of oxysterol metabolism [48]. These relationships underscore the role of both cholesterol and its oxidative products in atherogenesis, especially in pediatric patients, where early interventions are critical. Interestingly, in the healthy control group, the correlation between 27-OHC and 27-OHC/Total-C suggests that even in individuals without FH, the 27-OHC might serve as an early indicator of oxidative stress and cholesterol metabolism disturbances. This points to its potential as an early marker for atherosclerotic risk, warranting further investigation [48]. The lack of significant correlations between 5,6-α-epoxychol and oxidative stress markers or macrophage activation indicators suggests that this oxysterol may primarily reflect disruptions in lipid metabolism rather than generalized oxidative stress or inflammation. However, the significantly higher chitotriosidase and YKL-40 levels in the FH group indicate enhanced macrophage activation, a known contributor to atherosclerosis [49]. This dissociation suggests that oxysterol-driven atherogenesis may follow distinct or parallel pathways compared to macrophage-mediated inflammation. Finally, the increased CIMT observed in the FH group underscores the clinical relevance of these findings [50]. Elevated 5,6-α-epoxychol levels, along with their correlations with Total-C and LDL-C, suggest that monitoring oxysterol levels could provide valuable insights into the early progression of atherosclerosis in pediatric patients with FH.

The observation that the 27-OHC levels are higher in the healthy control group than in patients with FH may seem unexpected, given the established role of oxysterols in promoting atherogenesis [48]. However, this can be explained by the use of both atorvastatin and cholesterol-restrictive diets in FH management. Atorvastatin effectively lowers total cholesterol and oxysterol levels, including 27-OHC, by inhibiting key steps in cholesterol biosynthesis. Diet therapy, however, may have an even greater impact on 27-OHC levels, as studies have shown that high-cholesterol diets raise circulating 27-OHC and alter cholesterol metabolism [51, 52]. Therefore, the lower 27-OHC levels in patients with FH likely reflect the combined effects of these interventions. The correlation between 27-OHC and CIMT in patients with FH suggests that even residual levels of 27-OHC, despite treatment, contribute to atherogenesis. This relationship is further supported by the positive association with 5,6-α-epoxychol, another oxysterol linked to oxidative stress and endothelial dysfunction. The interactions between these oxysterols point to a complex relationship with atherosclerosis progression. The inverse correlation between C-Triol/Total-C and 27-OHC in patients with FH suggests distinct regulatory pathways, with C-Triol potentially being more responsive to statin therapy. Meanwhile, in healthy controls, the negative correlation between 27-OHC and age could indicate a natural decline in oxysterol levels due to reduced cholesterol turnover or oxidative stress. In conclusion, these findings highlight the differential effects of atorvastatin and diet therapy on oxysterols in patients with FH than in healthy individuals. Despite the benefits of statin therapy, the persistence of correlations with atherosclerosis markers like CIMT underscores the need for comprehensive strategies addressing both cholesterol and oxysterol metabolism to mitigate cardiovascular risks.

The significantly elevated levels of C-Triol and C-Triol/Total-C in the FH group compared to healthy controls emphasize the role of oxysterols in FH and atherogenesis. The C-Triol, a cholestane-triol derivative, is closely linked to cholesterol metabolism and oxidative stress. The stark difference in C-Triol levels between patients with FH and controls (P < 0.0001) suggests an increased oxysterol burden in patients with FH, driven by genetic predisposition and the hyperlipidemic state of FH [53]. The negative correlation between C-Triol and 27-OHC/Total-C in patients with FH may reflect compensatory adjustments in oxysterol metabolism, influenced by atorvastatin therapy and cholesterol-restrictive diets. Statins lower cholesterol synthesis, indirectly affecting oxysterol production, potentially explaining this inverse relationship [54]. The absence of correlations between C-Triol and oxidative stress markers or lipid profiles in patients with FH suggests that C-Triol may respond more to long-term statin therapy, influencing oxysterol ratios rather than absolute concentrations. In the control group, negative correlations between C-Triol, age, and PON1 (an antioxidant enzyme) suggest that C-Triol levels decrease with age and are inversely linked to antioxidant defenses. The negative relationship with 7-KC/Total-C further highlights metabolic differences in individuals without FH, potentially reflecting shifts in cholesterol metabolism with aging. Positive correlations between C-Triol, Total-C, and LDL-C in the control group align with the concept that higher cholesterol levels leads to increased oxysterol production. While this may not pose an immediate cardiovascular risk, it indicates the potential for oxysterol accumulation even in individuals without genetic hypercholesterolemia. The negative correlation between C-Triol/Total-C and PON1 in both the FH and control groups points to an interaction between oxidative stress and oxysterol metabolism [55]. Lower PON1 levels, indicative of reduced antioxidant capacity, may exacerbate the accumulation of oxysterols like C-Triol, fostering a pro-atherogenic environment. While statin therapy likely mitigates this interaction in patients with FH, the persistent elevation of C-Triol suggests that additional therapeutic strategies are needed to address oxysterol accumulation beyond cholesterol reduction alone.

The elevated levels of 7-Ketocholesterol (7-KC) in the FH group compared to healthy controls provide further evidence of increased oxidative stress and cholesterol oxidation in FH. As a non-enzymatic oxidation product, the 7-KC is known for its pro-inflammatory and pro-atherogenic effects [5]. The significant difference between the groups (P = 0.0047) suggests that despite atorvastatin therapy and cholesterol-restrictive diets, patients with FH continue to experience high oxidative stress, reflected in their elevated 7-KC levels. However, the lack of a significant difference in the 7-KC/Total-C between the groups indicates that cholesterol-lowering therapy reduces cholesterol levels but may not fully prevent oxysterol accumulation. This persistent oxidative burden underscores the complexity of managing FH and suggests that 7-KC could serve as a marker for residual cardiovascular risk. The positive correlation between 7-KC and 5,6-epoxychol/Total-C in the FH group highlights the interconnected role of these oxysterols in oxidative stress pathways contributing to atherosclerosis [56]. These findings suggest that, beyond lipid-lowering strategies, targeting oxidative processes may be essential to mitigate long-term cardiovascular risks in patients with FH. In the healthy group, the positive correlation between the 7-KC/Total-C and age suggests that the 7-KC levels increase with age, likely due to diminished antioxidant defenses or heightened oxidative stress. Negative correlations with catalase and C-Triol in the healthy group further emphasize the role of oxidative stress in oxysterol metabolism [57]. The study by Boenzi et al. (2016) on oxysterols in children with dyslipidemia, including FH, provides important insights into the role of 7-KC and C-Triol. While Boenzi et al. found normal oxysterol levels in their small cohort of three children with FH, the current study revealed significant elevations in both 7-KC and C-Triol. This discrepancy likely reflects differences in sample size and the severity of dyslipidemia and oxidative stress between the cohorts. These findings emphasize the potential of oxysterols as biomarkers for atherosclerosis progression in pediatric FH and highlight the need for further research to explore their utility in early atherosclerosis detection.

The CIMT demonstrated the highest AUC (0.99), confirming its strong discriminatory power between patients with FH and healthy controls, with high sensitivity (95.6%) and specificity (93.33%). This highlights CIMT’s reliability as a robust marker of atherosclerosis, especially in pediatric and adolescent patients with FH, where increased carotid thickness reflects early vascular changes due to hypercholesterolemia. The C-Triol also exhibited excellent diagnostic performance, with an AUC of 0.98, sensitivity of 95%, and specificity of 100%, making it one of the most promising oxysterol biomarkers for FH diagnosis. Its high accuracy suggests potential utility for monitoring disease progression and therapeutic response, given its strong links to oxidative stress and cholesterol metabolism. Similarly, the 27-OHC/Total-C achieved an AUC of 0.98, indicating its effectiveness in distinguishing patients with FH by reflecting cholesterol oxidation relative to total cholesterol. While 5,6-α-epoxychol displayed good discriminatory power (AUC = 0.83) with moderate sensitivity (85%) and specificity (80%), it appears less effective than C-Triol and CIMT for FH identification. The 27-OHC (AUC = 0.74) and 7-KC (AUC = 0.76) also showed moderate discrimination, with sensitivity and specificity values ranging from 65 to 80%. These markers provide some diagnostic value but are not as reliable as C-Triol or CIMT for identifying FH. Oxysterol-to-cholesterol ratios, such as 5,6-α-epoxychol/Total-C and 7-KC/Total-C, demonstrated weaker diagnostic power, particularly for 7-KC/Total-C (AUC = 0.50), which failed to distinguish between patients with FH and healthy controls. This suggests that absolute oxysterol levels, especially C-Triol, offer more valuable diagnostic information than their ratios to total cholesterol.

This study underscores the important role of oxysterols, oxidative stress, and macrophage activation in the development of atherosclerosis among pediatric and adolescent patients with FH. The elevated levels of C-Triol in patients with FH, compared to healthy controls, indicate significant disruptions in cholesterol metabolism and oxidative stress despite atorvastatin therapy. In contrast, the higher levels of 27-OHC observed in healthy controls suggest a differential regulation of oxysterols, potentially reflecting the effects of cholesterol-lowering treatments in patients with FH. While C-Triol was significantly elevated in patients with FH, the CIMT was also markedly increased, highlighting the early vascular changes associated with hypercholesterolemia. These findings suggest that C-Triol and 27-OHC/Total-C may serve as useful biomarkers for distinguishing patients with FH from healthy individuals. The study also emphasizes the need for therapeutic approaches targeting oxidative stress and macrophage activation, beyond conventional cholesterol-lowering therapies, to reduce cardiovascular risks in this population. Further research is required to explore the therapeutic potential of modulating oxysterol metabolism in patients with FH.

Study strengths and limitations

Strengths

This study contributes valuable insights into the relationship between oxysterol levels, oxidative stress, and atherosclerosis progression in children and adolescents with familial hypercholesterolemia (FH). It is one of the few studies to focus on pediatric and adolescent populations, a demographic that has been underrepresented in previous research on oxysterols and cardiovascular risk. The comprehensive approach of measuring multiple oxysterols—5,6-α-epoxychol, C-Triol, 7-KC, and 27-OHC—along with oxidative stress markers, macrophage activation indicators, and CIMT provides a multifaceted view of atherosclerosis development. Additionally, the use of the LC-MS/MS method ensures high sensitivity and specificity in oxysterol quantification. Another key strength is the strong discriminatory power of C-Triol and CIMT as biomarkers for distinguishing between FH patients and healthy controls, which suggests the potential for these markers to be used in early atherosclerosis detection and monitoring disease progression in clinical settings.

Limitations

The study has several limitations that should be considered when interpreting the findings. Firstly, the sample size is relatively small, with 20 patients with familial hypercholesterolemia and 20 healthy children and adolescents. This limited sample size may reduce the generalizability of the results, and larger studies would be needed to confirm these findings and increase statistical power. Secondly, the cross-sectional design of the study only provides a snapshot of oxysterol levels and related parameters at one point in time, limiting the ability to infer causal relationships or to observe changes over time. Thirdly, potential confounding factors such as diet, physical activity, or other lifestyle variables that could affect cholesterol levels, oxidative stress, or atherosclerosis risk were not controlled for in the study. Finally, the lack of follow-up data restricts the ability to assess long-term outcomes in children with hypercholesterolemia and whether changes in oxysterol levels correlate with the progression or regression of atherosclerosis over time.

Conclusion

This study highlights the pivotal roles of oxysterols, oxidative stress, and macrophage activation in the development of atherosclerosis among pediatric and adolescent patients with FH. Elevated levels of C-Triol, 5,6-α-epoxychol, and 7-KC in FH patients, alongside the significantly increased CIMT, underscore the early vascular changes associated with hypercholesterolemia. These findings suggest that oxysterols, particularly C-Triol, could serve as valuable biomarkers for monitoring atherosclerosis progression and assessing cardiovascular risk in FH patients.

From a clinical perspective, the identification of C-Triol and 27-OHC/Total-C as potential biomarkers provides clinicians with tools to better stratify cardiovascular risk in pediatric FH patients, even in those receiving statin therapy. Despite the benefits of cholesterol-lowering treatments like atorvastatin, the persistence of elevated oxysterol levels and their correlation with atherosclerosis markers indicates that additional therapeutic approaches targeting oxidative stress and macrophage activation may be necessary. Therefore, the clinical management of pediatric FH patients may benefit from incorporating oxysterol monitoring into routine care to allow for more personalized treatment strategies, aimed at mitigating cardiovascular risks at an early stage.

Furthermore, these findings support the need for future studies to explore therapeutic strategies that specifically address oxysterol metabolism and oxidative stress in addition to standard cholesterol-lowering treatments. Understanding the differential effects of oxysterol accumulation and its contribution to early atherosclerotic changes could lead to more effective interventions for preventing cardiovascular complications in FH patients, improving long-term outcomes and patient care.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

CHITO:

Chitotriosidase

CIMT:

Carotid intima media thickness

C-Triol:

(Cholestane-3β,5α,6β-triol)

DMAB:

4-(Dimethylamino)butyric acid

FH:

Familial hypercholesterolemia

HC:

Healthy control

HDL-C:

High-density lipoproteins cholesterol

LC-MS/MS:

Liquid chromatography tandem mass spectrometry

LDL-C:

Low-density lipoprotein cholesterol

MDA:

Malondialdehyde

NBT:

Nitroblue tetrazolium

PON1:

Paraoxonase 1

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

Total-C:

Total cholesterol

YKL-40:

Chitinase 3-like protein 1

4β-OHC:

4β-Hydroxycholesterol

5,6-α-epoxychol:

5,6-α-Epoxycholesterol ,

7-KC:

7-Ketocholesterol

7α-OHC:

7α-Hydroxycholesterol

7β-OHC:

7β-Hydroxycholesterol

24(S)-OHC:

24(S)-Hydroxycholesterol

27OHC:

27-Hydroxycholesterol

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Funding

This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) Project Number 121S335.

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Authors and Affiliations

  1. Department of Medical Biochemistry, Faculty of Medicine, Ege University, Bornova, Izmir, 35100, Turkey

    Erhan Canbay, Burak Durmaz, Oznur Copur, Begüm Tahhan & Ebru Sezer

  2. Department of Pediatric Metabolic Disease, Faculty of Medicine, Ege University, Bornova, Izmir, Turkey

    Ebru Canda, Havva Yazıcı, Sema Kalkan Ucar & Mahmut Coker

  3. Department of Pediatric Cardiology, Faculty of Medicine, Ege University Izmir, Bornova, Izmir, Turkey

    Gulcin Kayan Kasıkcı, Derya Aydın & Resit Erturk Levent

  4. Department of Biochemistry, Faculty of Science, Ege University, Bornova, Izmir, Turkey

    Dilek Düzgün & Zeynep Elçim Koru

  5. Department of Medical Biochemistry, Faculty of Medicine, Tınaztepe University, Buca, Izmir, Turkey

    Eser Yıldırım Sozmen

  6. Department of Medical Biochemistry, Faculty of Medicine, Near East University, Nicosia, Cyprus

    Burak Durmaz

  7. Department of Medical Pharmacology, Faculty of Medicine, Lokman Hekim University, Ankara, Türkiye

    Oznur Copur

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  1. Erhan Canbay
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Contributions

Erhan Canbay : Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft Ebru Canda : Investigation, Methodology Havva Yazıcı: Investigation Gulcin Kayan Kasıkcı : Investigation Burak Durmaz : Investigation Oznur Çopur : Investigation Begüm Tahhan: Investigation Dilek Düzgün: Investigation Zeynep Elçim Koru: Investigation Derya Aydın : Investigation Ebru Sezer : Writing - Review & Editing Ali Mert Özgönül : Investigation, Methodology Reşit Ertürk Levent : Supervision, Writing - Review & Editing Sema Kalkan Uçar : Supervision, Writing - Review & Editing, Methodology Mahmut Çoker : Conceptualization , Supervision, Writing - Review & Editing, Methodology Eser Sözmen : Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Supervision.

Corresponding author

Correspondence to Erhan Canbay.

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Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Ege University Faculty of Medicine (Ethics Committee Approval No: 20-8T/31). Written informed consent was obtained from the legal guardian, and the child provided assent. All methods were performed in accordance with relevant guidelines and regulations.

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Not applicable.

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The authors declare no competing interests.

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Canbay, E., Canda, E., Yazıcı, H. et al. Determination of selected oxysterol levels, oxidative stress, and macrophage activation indicators in children and adolescents with familial hypercholesterolemia. Lipids Health Dis 23, 374 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12944-024-02371-y

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12944-024-02371-y

Keywords

Lipids in Health and Disease

ISSN: 1476-511X