The Srb1+1050T Allele Is Associated with Metabolic Syndrome in Children but Not with Cholesteryl Ester Plasma Concentrations of High-Density Lipoprotein Subclasses

Abstract

Background:

Low cholesterol and phospholipid plasma levels of some high-density lipoprotein (HDL) subclasses have been described in children with metabolic syndrome. Scavenger receptor class B type I (SR-BI) has been proposed to be at the origin of such HDL alterations because of its key role on cholesteryl esters–HDL metabolism. However, the possible contribution of SR-BI has not been specifically explored in this kind of patients.

Methods:

Plasma lipid concentrations of HDL subclasses, i.e., triglycerides (TG), phosphatidylcholine (Ph), free cholesterol (FC), and total cholesterol (TC), were determined by enzymatic staining on polyacrylamide gradient gels (PAGE) in 39 pediatric patients with metabolic syndrome and 65 children as controls. Cholesteryl esters were estimated by the difference between TC and FC. Proteins of HDL subclasses were also stained for the assessment of the relative size distribution of HDL. For statistical analysis, the study population was grouped by Srb1 +1050C-->T polymorphism (rs5888) as carriers or noncarriers of the T allele, and data were corrected by metabolic syndrome status.

Results:

The Srb1 +1050T allele was associated with metabolic syndrome [odds ratio (OR)=2.18 (1.12–4.22), P=0.02]. Plasma TG corresponding to HDL3a, as well as the relative proportion of this HDL subclass, were slightly higher in carriers of the T allele as compared to CC homozygous subjects. Cholesteryl esters plasma concentrations of all HDL subclasses were comparable between T allele carriers and noncarriers after correction by metabolic syndrome status.

Conclusions

: Srb1 +1050T was associated with metabolic syndrome, but T carrier subjects did not show important differences concerning HDL subclasses as compared to noncarriers.

Introduction

S everal epidemiological studies have demonstrated a negative correlation between high-density lipoprotein cholesterol (HDL-C) and the development of coronary heart disease (CHD).^1,2 The causal relationship between plasma HDL-C concentration and CHD has been explained by the role played by these lipoproteins in reverse cholesterol transport, as well as by the potentially antiatherogenic properties of HDL, such as antiinflammatory, antioxidative, antiaggregatory, anticoagulant, and profibrinolytic effects.^2,3

The metabolic syndrome is defined as a concurrence of obesity, impaired glucose metabolism, hypertension, and dyslipidemia.^4
–6 Its prevalence has been increasing in children and adolescents.^7,8 Obesity and the closely linked insulin resistance (IR) are presumed to play a central role in the development of metabolic syndrome in pediatric patients,⁹ increasing the risk of future CHD or type 2 diabetes mellitus (T2DM) in adulthood.^10,11 In this context, the analysis of HDL subclasses may contribute to identifying more accurately those subjects at risk of T2DM and CHD, even from childhood.¹² HDL includes a heterogeneous group of lipoproteins that may be classified, by decreasing size, as HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c.¹³ These HDL subclasses have different antiatherogenic characteristics,^14
–16 but which HDL subfraction possesses the most important antiatherogenic role is still a matter of debate. It is likely that not only the size but also the lipid composition determine HDL functionality.^3,17
–19

A recent study from our laboratory²⁰ demonstrated that cholesterol plasma concentrations corresponding to the HDL2b, 2a, 3a, and 3b subclasses were decreased in children with metabolic syndrome. These results suggest a metabolic abnormality directly related to HDL size distribution and chemical composition. In this context, scavenger receptor class B type I (SR-BI) is of particular interest because of its active role in HDL remodeling²¹; SR-BI is a membrane glycoprotein receptor expressed in liver and steroidogenic tissues that promotes cholesterol ester uptake from plasma HDL into the liver.^21,22 Selective cholesterol ester uptake involves the preferential transfer of cholesterol ester from HDL into cells without accumulation or degradation of HDL apolipoproteins.²³ Large HDL, after interacting with SR-BI and selective cholesteryl esters uptake, become small HDL particles,²⁴ emphasizing the central role of this membrane protein in HDL remodeling and size distribution. In agreement with these results, the +1050C-->T polymorphism (exon 8 polymorphism, rs5888) of the Srb1 gene coding for SR-BI has been associated with HDL diameter in T2DM subjects, the T allele carriers having the largest HDL. However, little is known about the role of this polymorphism in HDL subclasses lipid composition and whether HDL size distribution is altered in stages previous to T2DM, i.e., the metabolic syndrome. Therefore, the aim of the present study was to explore the possible contribution of the Srb1 +1050C-->T polymorphism to HDL size distribution and the abnormal chemical composition of HDL subclasses in pediatric patients with MS previously reported.²⁰ Taking into account the SR-BI role in HDL metabolism, we estimated the cholesteryl esters plasma concentrations of the five HDL subclasses by a modification of an enzymatic method on semisolid phase.²⁵ Our results demonstrated an increased frequency of Srb1 +1050T allele in metabolic syndrome patients but a lack of association of lipid content with this polymorphism.

Methods

Study population

Volunteers for this study were recruited from two different centers, the clinical epidemiology research unit of the outpatient clinic IMSS (Mexico City, Mexico) and the outpatient nutrition clinic of the Children's Hospital (Toluca, Mexico). A total of 150 consecutive individuals, ages between 6 and 17 years, who agreed to participate in the study, were enrolled initially. After the explanation of the study details, 46 patients dropped out. The parents of 104 individuals gave their signed informed consent; 39 patients were diagnosed with metabolic syndrome based on the International Diabetes Federation (IDF) criteria for children and adolescents.²⁶ Sixty-five were healthy volunteers with a body mass index (BMI) between the 5^th and 85^th percentile (age and gender specific).²⁷ Measurements of blood pressure were obtained with standardized techniques. The protocol was approved by the Ethics Committees of the participant institutions.

Laboratory assessments

All patients were instructed to avoid strenuous exercise and to eat a light dinner the day before blood drawings were performed. Blood samples were obtained in EDTA tubes, after 12 h overnight fasting, from an antecubital vein after subjects had been seated for 15 min. Samples were centrifuged at 4°C, and plasma was separated and analyzed or frozen at −80°C until analysis. Plasma glucose, total cholesterol (TC), and triglycerides (TG) were determined by commercially available enzymatic methods. Phosphatidylcholine (Ph) and free cholesterol (FC) were determined by an enzymatic-colorimetric method (Wako Chemicals, USA). The phosphotungstic acid–Mg²⁺ method was used to precipitate apolipoprotein B (apoB)-containing lipoproteins before quantifying HDL–TC, HDL– FC, HDL–TG, and HDL–Ph plasma concentrations. Low-density lipoprotein cholesterol (LDL-C) was estimated in samples with TG less than 400 mg/dL.²⁸ Plasma levels of all lipids were determined within 24 h after drawing blood samples. Cholesteryl esters were calculated as the difference between TC and FC multiplied by a factor of 1.68.²⁹

Insulin was determined by a radioimmunoassay following manufacturer's instructions (Coat-A-Count, Diagnostic Product Corp., Los Angeles, CA); the inter- and intraassay variability coefficients were 2.1% and 6.8%, respectively. IR was estimated with the use of the homeostasis model assessment (HOMA-IR), as described elsewhere,³⁰ and was defined as a HOMA-IR value >2.01, which corresponds to the 75^th percentile found in a Mexican population sample of 881 adolescents (361 boys and 520 girls) with a mean age of 13.4±1.0.²⁰

Isolation of HDL

HDLs were separated by ultracentrifugation in a Beckman optima TLX table centrifuge at 100,000 rpm in 3.2-mL polycarbonate tubes and further separated by their hydrodynamic diameter by nondenaturing 3%–30% gradient polyacrylamide gel electrophoresis, as previously described.^13,29,31,32

Enzymatic lipid staining on polyacrylamide gels

Gels were stained for TC, Ph, and TG using enzymatic mixtures recently described.^20,25 Briefly, cholesterol esterase, cholesterol oxidase, and peroxidase at final concentrations of 0.075 U/mL, 0.05 U/mL, and 0.25 U/mL, respectively, were dissolved in a phosphate-buffered saline (PBS; 150 mM NaCl, 8.6 mM Na₂HPO₄, 1.4 mM NaH₂PO₄) (pH 7.4). The reaction mixture also included 3 mM sodium cholate, 0.1% Triton 100X, 0.4 mM thiazolyl blue tetrazolium bromide (MTT), 0.6 mM phenazine methosulfate (PMS), and carboxymethylcellulose at 1.4% as viscosant agent. For free-cholesterol staining, cholesterol esterase was omitted in the enzymatic mixture. The reactive mixture for Ph staining contained phospholipase D, choline oxidase, and peroxidase at final concentrations of 0.15 U/mL, 60 U/mL, and 0.25 U/mL, respectively, and 3 mM sodium cholate, 0.1% Triton 100X, 0.4 mM MTT, 0.6 mM PMS, and carboxymethylcellulose at 1.4% in PBS (pH 7.4). For TGs, the enzymatic mixture contained 40 U/mL lipase, 0.1 U/mL glycerol kinase, 0.4 U/mL glycerol 3-phosphate oxidase, and 0.25 U/mL of peroxidase in PBS. The mixture also contained 1 mmol/L MgCl₂ and 0.25 mmol/L adenosine triphosphate (ATP). Incubation times were between 60 and 75 min at 37°C in the dark for any lipid stained on electrophoresis gels.²⁰

At the end of the incubation time, the reaction mixture was removed and the gels were gently washed in PBS. Electrophoresis gels were then scanned in a GS-670 BioRad densitometer (scan 1), distained and further restained for proteins with Coomassie R-250, and scanned again (scan 2). The relative proportions of each HDL subclass determined per protein were estimated by optical densitometry analysis of scan 2, using as reference proteins (thyroglobulin, 17 nm; ferritin, 12.2 nm; catalase, 10.4 nm; lactate dehydrogenase, 8.2 nm; and albumin, 7.1 nm; high-molecular weight calibration kit, Amersham Pharmacia Biotech, Buckimghamshire, UK).^31
–33 The relative proportion of each HDL subclass is expressed as the percentage of the total HDL area under the curve, integrated from 7.94 to 13.59 nm.²⁵ For the classification of the HDL subclasses, we considered the following size intervals: HDL3c, 7.94–8.45 nm; HDL3b, 8.45–8.98 nm; HDL3a, 8.98–9.94 nm; HDL2a, 9.94–10.58 nm; and HDL2b, 10.58–13.59 nm [25].

TC, FC, TGs, or Ph plasma concentrations corresponding to each HDL subclass was estimated as follows: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes {10} {9} {7} {6} \begin{document} \begin{align*}{\rm HDL}n - {\rm L} = ({\rm HDL}n \times {\rm HDL} \hbox{-} {\rm L}) / 100\end{align*}\end{document}

where n represents the HDL subclass; %HDLn is the relative percentage of each subclass determined by optical densitometry, and L is the HDL-lipid (TC, free TGs, or Ph) plasma concentration determined by the commercial kits described above.^20,25 Cholesteryl esters plasma concentrations corresponding to HDL subclasses were estimated by the difference of HDL–TC and HDL–FC, multiplied by 1.68 to be expressed in milligrams per deciliter (mg/dL).

DNA extraction

Genomic DNA from whole blood containing EDTA was extracted by standard techniques.³⁴

Determination of the Srb1 genotypes

The Srb1 +1050C-->T (rs5888) single-nucleotide polymorphisms were genotyped using 5′ exonuclease TaqMan genotyping assays on an ABI Prism 7900 HT Fast Real Time PCR System, according to manufacturer's instructions (Applied Biosystems, Foster City, CA).

Statistical analysis

Gene frequencies of the Srb1 +1050C-->T polymorphism in the two groups were obtained by direct counting. Hardy–Weinberg equilibrium was evaluated by a chi-squared test. Differences in genotyping distribution were assessed by chi-squared analysis of the relevant 2×2 contingency table or the Fisher exact test, as appropriate. The P values were corrected (pC) according to the number of specificities tested and the number of comparisons performed, and they were considered statistically significant if their value were <0.05. Odds ratios (OR) with 95% confidence intervals (CI) were also calculated. Normal distribution of the variables was evaluated by the Kolmogorov–Smirnoff test. The significance of the differences between groups was tested by the Student t-test for normally distributed variables. Nonnormally distributed variables were logarithmically transformed for parametric statistical analysis. Comparisons of nonnormally distributed variables were performed by the Mann–Whitney U-test for independent groups. Partial correlations adjusted by age and gender were performed and statistical significance was set at P<0.05. Values are expressed as mean±standard deviation (SD) for variables with normal distribution and as median and interquartile interval for nonnormally distributed variables. Associations between metabolic syndrome and clinical risk factors including SR-BI polymorphisms were analyzed by binary logistic regression; results derived from the different tested models were expressed as OR and 95% CI for metabolic syndrome. The power of the study was based on changes of HDL size distribution as previously reported in pediatric patients with IR.¹⁷ Statistical analysis was performed using the SPSS v. 11 software.

Results

Table 1 shows mean clinical characteristics of the study sample. As expected, the parameters considered as inclusion criteria, such as blood pressure, fasting glucose, triglycerides, HDL-C, as well as waist circumference and BMI as indicators of obesity were different between controls and patients. The parameters related to glucose metabolism, including fasting insulin, glucose plasma levels, and HOMA-IR, were significantly higher in the metabolic syndrome group than in the control group.

Table 1.

Demographic, Anthropometric, and Biochemical Characteristics of the Individuals Included in the Study

	All subjects	Metabolic syndrome	Controls
n	104	39	65	P^a
Gender (female/male)	53/51	20/19	33/32	N.S.^b
Age (years)	11.2±3.1	11.2±2.8	11.2±3.3	0.947
BMI (kg/m²)	23.4±5.3	25.7±4.7	21.9±5.2	0.000
Waist circumference (cm)	77.3±11.6	82.9±10.2	71.4±10.0	0.000
SBP (mmHg)	98.0±6.8	102.1±7.3	93.6±5.8	0.005
DBP (mmHg)	62.7±4.7	65.6±4.1	59.5±3.5	0.004
Triglycerides (mg/dL)	109.5 [76.1–168.2]	170.0 [104.0–203.1]	96.1 [67.0–127.1]	0.000
Total cholesterol (mg/dL)	163.6±34.9	172.6±39.4	158.2±31.0	0.056
LDL-C (mg/dL)	95.1±30.2	102.3±36.0	90.9±25.4	0.087
HDL-C (total) (mg/dL)	41.3±10.4	32.5±6.8	45.4±11.0	0.000
HDL–free cholesterol (mg/dL)	11.9±4.6	10.0±3.2	13.4±5.0	0.000
HDL–esterified cholesterol (mg/dL)	47.0±14.5	41.6±10.3	51.2±15.9	0.001
HDL–triglycerides (mg/dL)	28.2±13.4	30.4±14.8	26.4±11.1	0.194
HDL–phosphatidylcholine (mg/dL)	115.9±41.1	110.4±32.5	120.3±46.7	0.258
Glucose (mg/dL)	90.7±13.12	98.3±10.3	86.2±12.6	0.000
Insulin (μU/mL)	9.5 [5.9–13.4]	10.9 [8.9–16.9]	6.7 [4.6–10.9]	0.000
HOMA-IR	2.65±1.76	3.37±2.02	1.90±1.00	0.000

Data are shown as mean±SD or median [interquartile range].

Metabolic syndrome vs. control group Student t-test or Mann–Whitney U test.

Chi-squared test.

BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance.

Allele and genotype frequencies of the Srb1 +1050C-->T gene polymorphism in metabolic syndrome patients and healthy controls are shown in Table 2. The observed and expected frequencies of genotype distributions were in Hardy–Weinberg equilibrium (P>0.05). Only two TT homozygous subjects were found in the metabolic syndrome group [genotype frequency (gf)=0.055], whereas there were 6 TT individuals in the control group [gf=0.092; P=not significant (N.S.)]. On the basis of such a low frequency of TT genotype and for comparison purposes, TT and TC subjects were integrated in a single group named “T carriers.” The T allele as well as T carriers were significantly more frequent in the metabolic syndrome group than in the control group (Table 2), suggesting a contribution of the Srb1 +1050C-->T polymorphism to the development of metabolic syndrome.

Table 2.

Allele and Genotype Frequencies of Srb1 +1050C–>T Polymorphism in Metabolic Syndrome Patients and Healthy Controls

Srb1 +1050C-->T	Metabolic syndrome (n=39)		Control (n=65)		pC	OR	95% CI
Allele	n	af	n	af
C	48	0.615	101	0.776	0.02	0.46	0.24–0.89
T	30	0.384	29	0.223	0.02	2.18	1.12–4.22
Genotype	n	gf	n	gf
TT+TC	26	0.666	23	0.353	0.002	3.65	1.46–9.24
CC	13	0.333	42	0.646	0.002	0.27	0.11–0.68

All populations were in Hardy–Weinberg equilibrium.

pC, P corrected values; OR, odds ratio; CI, confidence intervals; af, allele frequency; gf, genotype frequency.

Therefore, we looked further for a statistical association between the Srb1 +1050 T allele with metabolic syndrome by a binary logistic regression analysis (Table 3). Considering that BMI, plasma TGs, HDL-C, fasting glucose, and systolic or diastolic blood pressure were the factors that defined the metabolic syndrome [International Diabetes Federation (IDF) criteria²⁶], we first performed a logistic model that did not include these parameters (Table 3, model 1). In this initial model, we considered IR as an independent variable resistance because it could be considered the driving force of metabolic syndrome. Plasma cholesterol was also included in the model because this parameter tended to be higher in metabolic syndrome group (P=0.052; Table 1), as well as gender and age, considering that these parameters often contribute as confounding variables. Our results showed that only Srb1 +1050 T and IR predicted metabolic syndrome in this model. The inclusion of fasting insulin in the model did not modify these results (data not shown).

Table 3.

Binary Logistic Regression for Metabolic Syndrome in the Studied Population

Model 1	OR	95% CI	P
Insulin resistance	3.090	1.119–8.526	0.029
Srb1 +1050 T allele carrier	2.832	1.062–7.556	0.038
Model 2
Srb1 +1050 T allele carrier	4.283	1.033–17.768	0.045
Waist circumference	1.132	1.045–1.226	0.002
Triglycerides	1.026	1.006–1.045	0.009
HDL-C	0.816	0.725–0.918	0.001

The dependent variable in both models was metabolic syndrome.

Model 1 included as independent variables plasma cholesterol, insulin resistance, SR-BI T allele carrier, age, and gender.

Model 2 included as independent variables BMI, DBP, SBP, fasting insulin, glucose, plasma cholesterol, and SR-BI T allele carrier, age, and gender.

OR, odds ratio; CI, confidence interval; HDL-C, high-density lipoprotein cholesterol; BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure.

We also performed a logistic regression model including metabolic syndrome as dependent variable and parameters shown in Table 1 that were significantly different between groups: BMI, waist circumference, systolic and diastolic blood pressure, triglycerides, HDL-C, glucose, and fasting insulin (Table 3, model 2). We also included plasma cholesterol, age, and gender in this model. As shown in Table 3, the Srb1 +1050 T allele, TGs, HDL-C, and waist circumference were the parameters that predicted metabolic syndrome in the studied population. HOMA-IR was not included in this model because it was redundant with fasting glucose and insulin.

The focus of this study was to explore the possible contribution of the Srb1 +1050C-->T polymorphism to HDL structure; for this purpose, we compared HDL size distribution and HDL subclasses lipid concentrations in the studied population grouped by Srb1 +1050C-->T genotype (Table 4). As expected, a higher proportion of metabolic syndrome patients (53%) were included in the T carriers group as compared to the CC homozygous subjects group (23%, P<0.05). Consequently, statistical analysis was performed before and after correction by metabolic syndrome status. Total HDL-C (cholesteryl esters+free cholesterol) was lower in T carriers than in noncarriers (40.6±9.7 vs. 44.7±11.2 mg/dL, P<0.05), but this difference was not maintained after adjustment by metabolic syndrome status. Other components of the metabolic syndrome (systolic and diastolic blood pressure, waist circumference, and TGs) and IR were not different between T carriers and CC homozygous subjects (data not shown). It should be emphasized that the power of our study was particularly focused to observe changes on HDL size distribution.¹⁷ Therefore, the probability that we did not detect existing differences on HDL subclasses between T carriers and CC homozygous individuals was very low (<1%).

Table 4.

Relative Proportions Based on the Protein Content and Lipids Plasma Concentrations of HDL Subclasses of Srb1 +1050T Carriers (CT or TT genotypes) and CC Homozygous Individuals Included in the Study

	T carriers	CC homozygous	P^b	P corrected
n (metabolic syndrome/controls)	49 (26/23)	55 (13/42)^a
HDL subclasses (% of total protein)
HDL2b	14.3±3.7	16.0±6.9	0.164	0.819
HDL2a	10.0±1.7	10.0±2.3	0.940	0.305
HDL3a	27.4±3.3	25.7±2.9	0.017	0.023
HDL3b	21.5±1.9	20.9±2.3	0.183	0.594
HDL3c	26.9±6.3	27.5±8.6	0.729	0.221
Plasma concentrations (mg/dL)
Free cholesterol
Total HDL	10.8±3.1	12.9±5.5	0.027	0.249
HDL2b	2.6±0.9	3.4±1.7	0.022	0.255
HDL2a	1.3±0.4	1.6±0.7	0.026	0.266
HDL3a	2.8±0.9	3.3±1.4	0.059	0.347
HDL3b	1.7±0.6	2.1±0.9	0.038	0.188
HDL3c	2.2±1.0	2.5±1.3	0.253	0.410
Esterified cholesterol
Total HDL	44.5±12.8	49.1±15.6	0.141	0.601
HDL2b	12.2±4.3	14.3±6.7	0.092	0.462
HDL2a^c	5.5 [4.7-7.8]	5.4 [4.1-7.5]	0.632	0.245
HDL3a	12.6±3.9	12.7±4.7	0.979	0.513
HDL3b	6.5±2.2	7.8±3.3	0.047	0.213
HDL3c	6.2±3.8	8.0±3.9	0.040	0.071
Triglycerides
Total HDL	31.6±15.9	25.2±10.1	0.035	0.066
HDL2b	7.5±4.4	6.8±3.3	0.452	0.401
HDL2a	3.7±2.1	3.2±1.4	0.181	0.222
HDL3a^c	7.9 [5.2-10.8]	6.0 [4.2-8.5]	0.005	0.010
HDL3b	5.2±3.6	3.9±1.7	0.040	0.105
HDL3c	5.9±3.8	5.0±3.0	0.225	0.563
Phosphatidylcholine
Total HDL	114.9±35.4	116.9±45.9	0.823	0.951
HDL2b	27.9±12.9	34.1±18.8	0.086	0.349
HDL2a	13.4±4.9	15.0±6.4	0.209	0.596
HDL3a	30.2±9.3	29.7±11.9	0.841	0.651
HDL3b	19.1±5.9	18.0±7.7	0.483	0.432
HDL3c	21.2±8.2	19.3±9.7	0.347	0.573

Data represent mean±standard deviation (SD) for normally distributed variables or median [interquartile interval] for nonnormally distributed variables.

Chi-squared test <0.001.

Metabolic syndrome vs. controls, Student t-test, or Mann–Whitney U test for variables with normal or nonnormal distribution variables, respectively.

Logarithmically transformed for analysis.

HDL size distribution, determined by protein staining as indicated in the Methods section, was slightly different in T carriers; particularly, this group had a significant higher proportion of HDL3a subclass (Table 4). Such a slight difference remained significant after the adjustment by metabolic syndrome status. HDL-free cholesterol plasma concentrations and those corresponding to HDL2b, 2a, and 3b subclasses, as well as cholesterol esters from HDL3b and 3c, were significantly lower in T carriers than in the CC homozygous group. However, when data were adjusted by metabolic syndrome status, these differences were no longer statistically significant. HDL–TGs plasma concentrations as well as HDL3a TGs were higher in T carriers than in CC group, but only the latter remained significant after adjustment. Concerning HDL–Ph, HDL subclasses remained comparable between T carrier and CC homozygous groups (Table 4).

Discussion

In this study, we first determined the Srb1 +1050 genotype in pediatric patients with metabolic syndrome and control individuals. Our results demonstrated that the Srb1 +1050T allele was associated with metabolic syndrome pediatric individuals. To our knowledge, this is the first report that describes a higher frequency of this allele in this type of patient. This result seems to be consistent with the fact that Srb1 gene is located in the 12q24 region,³⁵ which has been postulated to harbor susceptibility genes for T2DM.^35

–38 However, it should be emphasized that the T allele was not associated with fasting insulin, glucose, HOMA, or waist circumference (data not shown). In agreement with this observation, Osgood et al. did not find a significant contribution of Srb1 +1050T to dyslipidemia in T2DM patients.³⁹ These data suggest that the higher frequency of the T allele in metabolic syndrome patients from our study is linked to other factors different from diabetes, i.e., the ethnicity. This hypothesis is consistent with the lower T allele frequency (af) observed in our study population as compared to previous reports; the T variant has been found to be higher in North American adults (af=0.486) than in our study (af=0.259). Moreover, in the former, there were no allele differences between DM2 patients and non-DM2 subjects. Therefore, it is likely that IR and T allele are two independent concurrent parameters in our metabolic syndrome patients, as suggested by the logistic regression analysis (model 1). We recognize that the number of individual included does not strengthen this study to reach solid conclusions about the allele frequencies; as a consequence, further studies with different cohorts are guaranteed to corroborate the ethnic factor as determinant of the higher allelic frequency of the T allele in Mexicans with metabolic syndrome.

Contrary to our expectations, the lipid profile from Srb1 +1050T carriers did not show any statistical difference when compared with noncarriers. In agreement with this result, Durst et al.⁴⁰ in a study including 56 Ashkenazi Jewish patients with familial hypercholesterolemia did not find any significant association between the Srb1 +1050 C-->T polymorphism and plasma cholesterol, HDL-C, LDL-C, or TGs. However, they observed higher HDL-C concentrations in carriers of the T allele in comparison with CC homozygous patients. Likewise, Plat and Mensink⁴¹ examined young men (n=41) and women (n=71), healthy and nonhypercholesterolemic Dutch individuals, and found a tendency that was nonstatistically significant to higher HDL-C plasma levels in carriers of the Srb1 +1050T allele as compared to homozygous for the C allele. Moreover, Osgood et al.³⁹ found a modest dose-dependent effect of the T allele; they observed higher plasma levels of HDL-C in TT (44.2 mg/dL) than in CC (42.1 mg/dL) homozygous North American subjects. In that study, T carriers had also lower levels of LDL-C, but this difference was only significant in women. Taken together, our results and those from other groups suggest that the Srb1 +1050T allele has just a modest, if any, influence on HDL-C plasma levels, probably also dependent on ethnicity.

We further investigated the influence of the Srb1 +1050T allele on the HDL structure. We hypothesized that T carriers should show altered HDL size distribution and HDL subclasses composition, particularly in cholesteryl esters content. The rationale for this idea was that, even if the Srb1 +1050 C-->T polymorphism results in a synonymous nucleotide substitution, it has been recently demonstrated that this variant drives to a lower SR-BI protein expression and function.⁴² In this work, Constantineau et al., using several in vitro models, found that the rs5888 polymorphism affects the SR-BI RNA secondary structure, which changed its ability to undergo productive protein translation, thus lowering SR-BI protein expression. Moreover, low SR-BI function has been associated to high HDL-C plasma levels,^43,44 because cholesteryl esters could not be cleared from these lipoproteins. Therefore, we expected to find an HDL size distribution shifted toward large HDL particles and a high cholesteyl ester content in large HDL subclasses.

For the assessment of cholesteryl esters from HDL subclasses, we developed a new procedure based on a modification of a previously reported enzymatic method in semisolid phase.²⁵ In a preliminary analysis, we observed several differences between groups concerning HDL subclasses profile, particularly a low plasma concentration of cholesteryl esters of small HDL3b and 3c. However, most of these differences lost significance when we adjusted by metabolic syndrome status. These results confirm our previous report indicating that metabolic syndrome is strongly associated with HDL subclasses' abnormalities²⁰ and demonstrate that the Srb1 +1050T allele has a very limited effect on HDL size distribution and lipid content. Recently, Naj et al. reported an association of the Srb1 rs10846744 polymorphism with subclinical atherosclerosis in African-American, European American, and Hispanic individuals.⁴⁵ This association was independent of lipids and other cardiovascular risk factors. In our work, it was not possible to determine this polymorphism, however, according to the HapMap data, the rs5888 and the rs108467 are not in linkage disequilibrium. In summary, the Srb1 +1050T allele is significantly more frequent in Mexican pediatric patients with metabolic syndrome as compared to control children, but we did not find any important effect of this allele on plasma lipid levels, HDL size distribution, and HDL subclasses lipid content.

Footnotes

Acknowledgments

This study is CONACYT Project 132473.

Author Disclosure Statement

No competing financial interests exist.

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