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Oxysterol as a Marker of Atherogenic Dyslipidemia in Adolescence

School of Dietetics and Human Nutrition (D.A., G.E., S.K.), McGill University, Québec, Canada H9X 3V9; Laboratory of Natural Medicinal Compounds (J.V.), Migal-Galilee Technology Center, Kiryat Shmona 11016, Israel; and Department of Medicine (S.M.), Division of Endocrinology and Metabolism, McGill University Health Center, Royal Victoria Hospital, Montréal, Québec, Canada H3H 2R9

Address all correspondence and requests for reprints to: Dr. Stan Kubow, School of Dietetics and Human Nutrition, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada. E-mail: stan.kubow{at}mcgill.ca.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Oxysterols represent potentially important oxidative stress biomarkers in adolescence.

Objective: The objective of the study was to examine the relationship between the concentrations of serum enzymatically and nonenzymatically generated oxysterols, measures of obesity, and metabolic components including insulin resistance and levels of blood pressure and serum lipids.

Design: This was a cross-sectional study.

Setting: All subjects were examined between 2003 and 2005 at a hospital, a part of a follow-up evaluation mother-daughter pairs representing pregnancies affected or unaffected by gestational diabetes that resulted in the deliveries in 1989–1991.

Subjects: Subjects included a subset (n = 89) of the total study population of 189 adolescent girls with a mean age of 15.32 ± 0.65 yr and body mass index of 22.54 ± 3.98 kg/m².

Main Outcome Measures: Measures included serum levels of the oxysterols 7-hydroxy-cholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol; and body mass index, homeostasis model assessment insulin resistance index, fasting insulin, fasting glucose, blood pressure, total cholesterol, non-high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, and apolipoprotein B (ApoB).

Results: Serum oxysterol concentrations in the adolescent cohort correlated positively with insulin (P < 0.05), total cholesterol (P < 0.05), non-high-density lipoprotein cholesterol (P < 0.05), low-density lipoprotein cholesterol (P < 0.05), and ApoB (P < 0.01). ApoB and fasting insulin were found to be the major determinants of serum oxysterols after adjustment for body mass index. Being a daughter of gestational diabetes pregnancy alone did not seem to be a predisposing factor to increased oxidative stress in our cohort.

Conclusion: Serum oxysterol concentrations increase with obesity, insulin, and ApoB, which are established derangements associated with the metabolic syndrome.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Oxidative stress is hypothesized to be a potential link in metabolic disturbances such as insulin resistance, hypertension, and dyslipidemia seen in high-risk groups for type 2 diabetes (T2DM), the metabolic syndrome, and cardiovascular disease (CVD) (1). Numerous animal and in vitro studies have shown consistently that oxidative stress can influence directly mechanisms involved in β-cell dysfunction, insulin action, and endothelial dysfunction and thus may be implicated in their etiology (2, 3).

In clinical studies, free radical-generated oxysterols have been reported to be elevated in specific chronic disease populations that include patients with diabetes, hypercholesterolemia, and advanced carotid atherosclerosis and individuals at increased risk for CVD (4, 5, 6). Oxysterols are the derivatives of enzymatic or nonenzymatic cholesterol oxidation, and they may be involved in the initiation and progression of atherosclerosis (7). Many studies have reported the presence of different types of predominantly free radical-induced oxysterols in human lipoproteins, femoral plaques, and atherosclerotic aorta (4, 8, 9). Conversely, enzymatically generated oxysterols are indicated to play a role in the regulation of cholesterol metabolism (10) and act as important modulators of insulin by acting as agonists of liver X receptors (LXRs) (10). The assessment of plasma or serum oxysterols as an index of oxidative stress using gas chromatography-mass spectrometry (GC-MS) is an excellent approach due to its high selectivity.

This cross-sectional study was performed to assess serum oxysterol concentrations in a cohort of teenage girls. This study is part of a more comprehensive ongoing follow-up evaluation of early modifiable T2DM risk factors in mother-daughter pairs representing pregnancies affected and unaffected by gestational diabetes mellitus (GDM) from term deliveries at the Royal Victoria Hospital between 1989 and 1990. Women with prior history of gestational diabetes as well as their offspring are considered a high-risk group for the development of T2DM and the metabolic syndrome, both of which are considered major risk factors of CVD (11). The overall aim was to investigate oxidative stress status in the cohort based on different risk categories including mother’s pregnancy status (GDM vs. non-GDM) and daughters’ obesity. The study also examined the relationship between serum oxysterol concentrations and measures of obesity, measures of glucose tolerance, and metabolic components that include levels of blood pressure and serum lipids.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subject recruitment

Participants were a subsample taken from a comprehensive follow-up evaluation of early modifiable T2DM risk factors in mother-daughter pairs representing pregnancies affected and unaffected by GDM from term deliveries at the Royal Victoria Hospital between 1989 and 1990. A detailed description of the mother-daughter study protocol will be published elsewhere. Daughters were evaluated for their dietary habits, physical activity, psychosocial factors, medical history, blood pressure, anthropometric indices, fasting lipids, and various clinical indices including glucose and insulin during a 75-g glucose load. Percent body fat was assessed via foot to foot bioelectrical impedance analysis (BF350; Tanita, Tokyo, Japan). Fasting blood samples were prepared and stored at –80 C for future analyses. The study was approved by the Ethical Review Board of the McGill University Health Center, and informed consent was obtained from all participants.

Subject characteristics

This study involved the first 94 daughters from the mother-daughter cohort study conducted at the Royal Victoria Hospital. After excluding self-reported smokers (n = 5) from the current study, there were 45 cases identified to be daughters of GDM pregnancies. The large majority of subjects were Caucasian (77.5%, self-reported). The mean age of the subjects was 15.30 ± 0.66 yr, and all were menstruating; therefore, metabolically they are considered as young adults. Subjects reported no serious health problem; however, 13 subjects reported being asthmatic. In addition to asthma-related medications, subjects reported taken antibiotics (n = 5); central nervous system medications such as antianxiety, anticonvulsant, antidepressant (n = 4); contraceptives (n = 5); and antihistamines (n = 5). Participants were also taking over-the-counter medications such as Advil and/or Tylenol (n = 7) as well as dietary supplements (n = 17).

Procedure

Extraction and analysis of serum cholesterol and oxysterols. To each serum sample (200 µl), 19-hydroxycholesterol was added (50 µl 19-hydroxycholesterol from solution of 50 µg/ml) as internal standard and gently vortexed. For oxysterol extraction, 0.1 g NaCl was added to the serum sample, and the mixture was vortexed for 20 sec. The serum sample was extracted by addition of 2.5 ml 0.01% butylated hydroxytoluene hexane/2-propanol [3:2 (vol/vol)] solution and then vortexed for 1 min. After centrifugation at 1877 x g at 4 C for 4 min, the upper organic layer was collected and the extraction procedure was repeated with additional 2.5 ml 0.01% BHT hexane/2-propanol [3:2 (vol/vol)] solution, vortexed, and centrifuged at 1877 x g at 4 C for 4 min. The organic phases were combined and 0.2 g anhydrous Na₂SO₄ was added. The solution was vortexed for 20 sec and allowed to stand for 15 min at room temperature. The solution was filtered using glass wool inserted into small Pasteur pipettes. The organic phase (4–5 ml) was evaporated under N₂ purging and the residue stored at –80 C.

Samples for oxysterol and cholesterol analysis were processed according to the method of Vaya et al. (8). Briefly, the dry residue of the extracted sample was dissolved in 1 ml diethyl ether, and 1 ml 20% KOH in methanol (wt/vol) was added. The remaining head space of the vial was filled with nitrogen and the reaction mixture was left in the dark at ambient temperature for 3 h. The mixture was then neutralized by adding 1 ml 25% citric acid in water, 2 ml diethyl ether was added and vortexed, and the upper organic phase was collected. The remaining aqueous layer was washed two more times with 1.5 ml diethyl ether, and the organic layers were collected, combined, dried (sodium sulfate), filtered, and evaporated to dryness under nitrogen.

Detection of serum oxysterols by GC-MS

Standards or the dried extracts were subjected to a silylating reagent, N,O-bis(thrimethylsilyl) acetamide (BSA; 100 µl), followed by the addition of 1,4-dioxane (dried on 4Å molecular sieves and passed through aluminum oxide; 100 µl) and heated to 70 C for 30 min. Samples were analyzed on GC-MS coupled to a HP 5972 quadrupole mass spectrometer and linked to a HP ChemStation data system (Hewlett Packard, Portland, OR). The gas chromatograph was fitted with a 30-m HP-5 trace analysis capillary column (0.32 mm inner diameter, 0.25 µm film thickness, 5% phenylmethyl silicone) and operated in splitless mode for 0.8 min and then in split ratio of 1:1. Helium was used as carrier gas at a flow rate of 0.656 ml/min, pressure 10.4 pounds per square inch, and at a linear velocity of 31 cm/sec. The mass spectrometry transfer line was maintained at 280 C. The injector was set at 300 C; the detector at 330 C; and the column heated at a gradient, starting at 200 C, increasing to 250 C at 10 C/min and then at 5 C/min to 300 C, and maintained for an additional 15 min at 300 C. Samples were detected in the GC-MS in total ion monitor from which two to four most representative ions were selected for reinjection in single ion monitoring mode. The mean quantity of each oxysterol was calculated from calibration curves of its standards. Under the above conditions, the limit of detection for each oxysterol was determined with deviations of less than 6% of the mean. Corresponding areas equal to 10 times the area measured in the blanks were set as the limit of detection. 19-hydroxycholesterol was used as the internal standard. In each sample, serum levels of the oxysterols 7-hydroxycholesterol (7OH), 7β-hydroxycholesterol (7βOH), 5,6 β-epoxycholesterol, 5,6 -epoxycholesterol, 7-ketocholesterol (7-keto), and 3,5,6-trihydroxycholesterol were evaluated. The selection of oxysterols was based on previous literature, and the oxysterols were more likely obtained under in vivo cholesterol oxidation. The detection limit via this method for the oxysterols measured ranges from 0.06 to 0.12 ng/injection.

Validation of the analysis

A standard of cholesteryl linoleate was subjected to identical saponification and extraction conditions, and cholesterol was recovered with a 98% (± 5%) yield. The stability of the oxysterols under the experimental conditions was also verified (extraction, hydrolysis, reextraction, and silylation). Thus, β-epoxy, 7-keto, and cholestane-3,5,6-triol were subjected to saponification under identical conditions after extraction and GC-MS analysis, using the external standard method. The recovery was almost quantitative (relative standard deviation ± 4.6–6.8%). Silylation with BSA in 1,4-dioxane was found to be milder and superior, in terms of reaction conversion, reproducibility, and suitability, to GC-MS to other reagents examined, such as trimethylsilylchloride in pyridine and BSA in other solvents (dimethylformamide, diethyl ether).

Statistical analysis

Anthropometric, clinical, and biochemical measures for all the subjects were reported as mean ± SD. All variables were treated on a continuous scale in statistical analyses. Variables that failed the normality test were logarithmically transformed before the analysis to allow for assessment via parametric tests. For log-transformed data, results are given as geometric mean ± SD. The serum levels of oxysterols were compared between cases and controls by Student’s t test. In addition, serum levels of oxysterols were compared using Student’s t test between different categories, i.e. medication vs. nonmedication use, obese vs. nonobese, and insulin resistant vs. noninsulin resistant. Correlation analysis was performed using Pearson’s correlation analysis to assess the relationship between serum oxysterol concentrations and both lipoprotein parameters and blood pressure. All P values were two tailed, and P < 0.05 was considered significant for all tests performed.

To estimate the final predictors of oxysterol variability and examine the influence of confounding variables, multivariable analysis with stepwise regression was used. For the stepwise regression, an -value of 0.05 was used to exclude variables that had little or no influence on the oxysterol under analysis. Statistical analyses with oxysterol values were performed corrected by the internal standard used in addition to with and without correction for total cholesterol content. Body mass index (BMI) percentiles were calculated using Epiinfo version 3.3.2 [Centers for Disease Control and Prevention (CDC), Atlanta, GA]. All statistical analysis was performed using SPSS version 13.0 software (SPSS Inc., Chicago, IL).

Definitions Obesity was defined using both The CDC’s BMI percentile for age established cutoffs (12, 13) and the International Obesity Task Force (IOTF) age-specific BMI cutoffs (14, 15). Total cholesterol and low-density lipoprotein cholesterol (LDL-C) are identified as early cardiovascular risk factors and are used for the diagnostic criteria set by the U.S. National Cholesterol Education Program for adolescents (16). Subjects were classified according to the American Diabetes Association criteria for impaired glucose tolerance and T2DM (17).

Calculations Subjects were identified as insulin resistant if their homeostasis model assessment insulin resistance index (HOMA-IR) value was 3.16 or greater according to Keskin et al. (18); whereby ROC validated level for HOMA-IR diagnostic cutoff for insulin resistance in adolescents was established. Non-high-density lipoprotein (HDL) cholesterol was calculated as total cholesterol minus HDL cholesterol, which provides a single index of all the atherogenic, apolipoprotein (Apo)-B-containing lipoproteins-low-density lipoprotein (LDL), very low-density lipoprotein, intermediate-density lipoprotein, and lipoprotein(a) (19).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Anthropometric and clinical indices

The study sample characteristics are presented in Table 1. With regard to the adiposity indices, the mean weight in kilograms was 59.32 ± 12.36. The BMI percentile was 62.19 ± 27.28. According to the CDC criteria, eight subjects were identified to be obese, 19 subjects were overweight, and 67 subjects were normal weight. The mean levels of all of lipid components were in the normal range (Table 1). However, seven subjects had elevated total cholesterol (5.18 mmol/liter), 12 subjects had elevated LDL cholesterol (3.36), eight subjects had elevated triglycerides (1.243), and 24 subjects showed low HDL cholesterol (1.036). None of the subjects were identified to have blood pressure abnormalities or as cases of either impaired glucose tolerance or impaired fasting glucose. Using HOMA-IR cutoff value (3.16), 16 subjects were identified as insulin resistant.

The six different oxysterols that were identified in the serum of subjects included 7OH, 7βOH, 5,6β-epoxycholesterol, 5,6-epoxycholesterol, 7-keto, and 3,5,6-trihydroxycholesterol. For the ease of interpretation and comparison and based on previous literature findings, the analysis focused mainly on the 7-oxysterols, which are the products of in vivo autooxidation of cholesterol (7βOH and 7-keto). The data on oxysterols were not normally distributed. Therefore, all data were transformed to the natural logarithm scale to create distributions that are closer to normal distribution.

The mean level ± SD (range) for all 89 participants was 153.85 ± 1.55 (72.44–831.76) for 7OH, 88.57 ± 1.74 (30.90–501.19) for 7βOH, and 796.16 ± 1.79 (223.87–5623.41) for 7-keto (Table 2). In terms of the proportion of each oxysterol in the sample, 7-keto levels were generated the highest (76.06 ± 5.75%) compared with 7OH (15.26 ± 4.50%) and 7βOH (8.67 ± 2.06%). For all statistical analyses, mean ± SD were based on log scale values and transformed back to original units corresponding to the geometric mean for each category.

The basal clinical and biological characteristics of the different categorizations are reported in Table 1, and the comparison of serum oxysterol concentrations among the categories are reported in Table 2. Only obesity categorization reached statistical significance, even after adjustment of total cholesterol. Overweight/obese subjects, identified with either CDC (n = 24) or IOTF (n = 27) criteria, had higher mean levels of all the serum oxysterols each individually or combined as total oxysterols (Table 2). In addition to obesity categorization, the difference in mean concentrations of 7βOH was found significant (76.95 ± 1.51 vs. 96.63 ± 1.85, P = 0.05) in medication takers (Table 2), and the difference remained significant when values were adjusted for total cholesterol for 7βOH (48.14 ± 1.36 vs.65.09 ± 1.41, P = 0.035) and 7OH (26.16 ± 1.48 vs.33.47 ± 1.41, P = 0.016).

Correlates of serum oxysterols

All oxysterols behaved similarly in terms of the correlation analyses, with few exceptions (Table 3). The oxysterols, 7OH, 7βOH, and 7-keto, both separately and combined, correlated positively with measures of obesity [waist circumference (WC) and BMI]. In addition, all oxysterols correlated positively with total cholesterol, LDL cholesterol, ApoB, and non-HDL cholesterol. Both 7OH and 7βOH correlated significantly with fasting insulin (r = 0.283 and r = 0.218, respectively), and only 7OH correlated positively with HOMA-IR (r = 0.214). When oxysterol concentrations were corrected for total cholesterol, all relationship disappeared with the exception of 7OH, with both correlations to insulin (r = 0.278) and HOMA-IR (r = 0.281) remaining significant.

The results of the stepwise multivariable regression models are contained in Tables 5 and 6. The variables used in our models were highly intercorrelated (data not shown). Many models were generated to avoid this collinearity; however, all arrived at the same findings reported. When serum oxysterol concentrations were not adjusted for total cholesterol, ApoB was found to be the determinant of serum oxysterol concentrations [7-keto and Total-7-oxysterols (Tot-7-Oxy), R² = 0.072, and 7βOH, R² = 0.116], with the exception of 7OH, for which both fasting insulin and ApoB were its determinants (R² = 0.157). When serum oxysterols were corrected for total cholesterol, fasting insulin was the only predictor that remained in the models for both 7OH (R² = 0.081) and 7βOH (R² = 0.045).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The positive relationship of insulin with the enzymatically generated oxysterol 7OH seen in the present study is supported by previous research showing such oxysterols can act as important modulators of insulin (31). Enzymatically produced oxysterols are established agonists of LXRs, which are involved in increasing insulin secretion in addition to their known role in the regulation of cholesterol metabolism (10). Previous work has demonstrated that 7OH, which originates mostly from the hepatic CYP7A1 activity, is an important ligand of LXRs (32). A positive relationship between insulin and the serum oxysterol 7OH was observed to persist after controlling for non-HDL cholesterol and BMI (Table 4). Thus, our findings suggest that 7OH could affect insulin secretion, which might be mediated by its putative role as an LXR agonist. Additionally, insulin was shown to be a 7OH predictor in the regression model (Tables 5 and 6), which corresponds to previous literature showing that insulin at physiological concentrations induces CYPA1 activity (31). To our knowledge, the above findings present the first in vivo evidence capturing the oxysterol link to insulin status (Table 6). The detection of these oxysterol-insulin relationships in our study might have been more apparent in this healthy cohort because it is unaffected by the confounding effect of the clustering of deranged metabolic abnormalities associated with the insulin-resistant state (data not shown).

An early metabolic derangement of the metabolic syndrome in the form of dyslipidemia was present in our cohort. In contrast to many previous studies, serum oxysterol concentrations either separately or the combined total, were correlated positively with total cholesterol (P < 0.05), LDL-C (P < 0.05), ApoB (P < 0.01), and collectively with non-HDL cholesterol (P < 0.05) (Table 3). Correlations between oxysterol concentrations and absolute blood lipid concentrations have been contradictory. Abo et al. (33) found a positive association between plasma 7-keto and serum cholesterol concentrations in poorly controlled diabetics and controls. In contrast, Iuliano et al. (34) in healthy Italian volunteers demonstrated that neither 7-keto nor 7βOH correlated with total cholesterol concentrations, thereby indicating that oxysterol production is not necessarily related to cholesterol increased availability. The absence of confounding influences in the early stage of metabolic syndrome in our adolescent cohort could be responsible for the observed linear relationship of serum oxysterol concentrations with total cholesterol, LDL-C, and ApoB (Table 3). Hence, the present findings indicate that oxysterol concentrations are related to the presence of the early stage metabolic syndrome in adolescent girls, which always involves high cholesterol levels as a major component.

We determined that ApoB is a direct predictor of serum oxysterols and even after adjustment for both total cholesterol and non-HDL cholesterol (Table 4). These latter relationships are not surprising because ApoB is the primary site at which the oxidation of cholesterol and protein in LDL occurs (35). In that regard, a strong positive relationship between 7-keto and Tot-7-oxy concentrations with ApoB concentrations was observed that persisted throughout all the statistical analyses (Table 4). The latter findings are consistent with evidence for enhanced oxidative stress to LDL-C that is associated with the metabolic syndrome (25). In that regard, small dense LDL is a standard component of the dyslipidemia in the metabolic syndrome, which is associated with a greater susceptibility to oxidative stress (25). It is noteworthy that ApoB is associated more closely with inflammatory markers and insulin resistance than triglycerides and all the cholesterol markers (36). Our finding that oxysterols associated with ApoB emphasizes the potential versatility of the role of oxysterols as a marker of atherogenic dyslipidemia. Taken together, the results imply that oxysterol formation could be important link between hypercholesterolemia and metabolic derangements observed in the progression to vascular dysfunctions.

Important components of the metabolic syndrome such as HDL cholesterol and triglycerides did not correlate with serum oxysterols, which could be related to confounding lifestyle factors like physical activity not accounted for in our study. The relationship between HDL cholesterol and serum oxysterol concentrations might have been obscured by the previously characterized antioxidative and prooxidative outcomes of HDL cholesterol that might occur simultaneously at this early metabolic stage (22). The lack of relationship found between serum oxysterol concentrations and serum triglycerides, HDL cholesterol, or blood pressure is also likely due to the lack of clustering of these risk factors in the young cohort. Furthermore, the small subject number of this study may not have provided statistical power to detect differences in serum oxysterol concentrations between the insulin-resistant group and counterpart as expected from the direct correlations with HOMA-IR observed in this study (Tables 3 and 4).

Many factors cause variation in tissue oxysterol content, including medication intake, smoking, over-the-counter antioxidant supplement use and other dietary factors (37, 38, 39, 40). When oxysterol values were analyzed in terms of potential covariates, medication use was clearly associated with the lowest serum oxysterol concentrations despite evident metabolic disturbances in lipid profiles among the medication users (Table 2). Most individuals consumed either antiinflammatory or antibiotic medications, which might have exerted antioxidant effects. Unfortunately, the cross-sectional study design and the relatively small sample size precluded more detailed statistical analyses to adjust for these significant covariates for oxysterol concentrations.

In summary, this study presented a unique opportunity to assess the development of certain risk factors of T2DM and the metabolic syndrome at an early stage. To our knowledge, this is the first study examining serum oxysterol concentrations in adolescence as a potential indicator for assessing certain metabolic derangement such as dyslipidemias associated with T2DM and sequeale. The present findings in our adolescent cohort suggest that oxysterols are present as early markers of oxidative stress-mediated dysregulations involving cholesterol metabolism and thus be a metabolic link to the clustering of vascular risk factors in adolescence.

	Acknowledgments

 
We acknowledge the assistance of the mothers-daughters study staff at the Royal Victoria Hospital, Ms. Claire Gougeon, Mrs. Maria Nudi, and Ms. Sina Gallo for their help with interviews and coordination of the study, and we thank all participants of the study.

	Footnotes

 
This work was supported by a grant from the Canadian Institutes of Health Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 19, 2008

Abbreviations: Apo, Apolipoprotein; BMI, body mass index; BSA, bis(thrimethylsilyl) acetamide; CVD, cardiovascular disease; GC-MS, gas chromatography-mass spectrometry; GDM, gestational diabetes mellitus; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment insulin resistance index; IOTF, International Obesity Task Force; 7-keto, 7-ketocholesterol; LDL, low-density lipoprotein; LDL-C, LDL cholesterol; LXR, liver X receptor; 7OH, 7β-hydroxycholesterol; 7βOH, 7-hydroxycholesterol; T2DM, type 2 diabetes; WC, waist circumference.

Received March 13, 2008.

Accepted August 8, 2008.

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