Soluble lectin-like oxidized low density lipoprotein receptor-1 in metabolic syndrome

A case-control study was conducted at Thammasat University Hospital, Thailand. A total of 1,100 participants, Thammasat University officers who presented for an annual checkup in January 2009, were retrospectively enrolled and their demographic data (sex, age, weight, height, and blood pressure) were recorded. We selected 148 cases of metabolic syndrome and 206 normal controls using the National Cholesterol Educational Program Adult Treatment Panel III (NCEP-ATP III) criteria with a modification, replacing waist circumference with body mass index (BMI) [20, 21]. According to the modified NCEP-ATP III guidelines, metabolic syndrome is diagnosed when three or more of the following risk factors are present: (1) BMI ≥25 kg/m² for women and ≥27 kg/ m² for men, (2) fasting plasma glucose (FPG) ≥110 mg/dL, (3) systolic blood pressure ≥130 mmHg or diastolic blood pressure ≥85 mmHg, (4) fasting triglyceride ≥150 mg/dL, and (5) high-density lipoprotein cholesterol (HDL cholesterol) <40 mg/dL in men and <50 mg/dL in women. For control participants without metabolic syndrome, none above five risk factors were presented. The research protocol was reviewed and approved by the Human Research Ethics Committee of Thammasat University (No. 40/52). The Ethics Committee specifically waived the usual requirement for informed consent provided patient confidentiality was protected according to the principles of the contemporary version of the Declaration of Helsinki, and permitted demographic data collection and the reuse of samples taken for clinical purposes at the annual checkup in January, 2009.

Fasting blood samples of all participants were drawn and used for determining the classical risk factors: FPG, total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglyceride using automated blood analyzer (Dimension clinical chemistry; RxL Max, Siemens).

Serum sLOX-1 was analyzed using sandwich enzyme-linked immunosorbent assay (DY1798, R&D system) and ELISA plate reader (Opsys MR, Dynex). Briefly, in each well of the microplate mouse was coated with anti-human LOX-1 (2 μg/mL). After blocking nonspecific binding sites with 1% bovine serum albumin in phosphate-buffered saline (PBS) diluent, test sample or standard (recombinant human LOX-1) dissolved in diluent (31-2000 pg/mL) were added (0.1 mL per well). After incubation for 2 hours at room temperature, the well was washed with 0.05% Tween 20 in PBS, pH 7.4. The sLOX-1 and recombinant human LOX-1 retained on the wells were detected by adding biotinylated goat anti-human LOX-1, streptavidin conjugated to horseradish-peroxidase, and tetramethyl benzidine substrate. The color reaction was determined using microplate reader at λ_450nm, subtracted with the reading at λ_570nm. The coefficient of variation was 3.6% (n = 10) and the percent recovery was103.3% (n = 10). Sensitivity and specificity of sLOX-1 were analyzed using a receiver operating characteristic (ROC) curve.

Results are expressed as means and standard deviation (SD) or as median and interquartile range if the distribution of the data is skewed. Comparisons were performed using a chi-square or Fisher exact test for proportion, and a Student t or Mann–Whiney U test for continuous variables. The level of significance was set at P < 0.05. Spearman’s rank correlation coefficients were used to examine the correlation between sLOX-1 levels and age, BMI, blood pressure, FPG, and lipid profiles with a significance cut off at P < 0.001. Multiple linear regression analysis was used to evaluate the contribution of each confounding factor for sLOX-1. Univariate and multivariate analysis were conducted using a logistic regression with 95% confidence interval. The Statistical Package for the Social Sciences software (version 16, SPSS, Chicago, IL, USA) was used for statistical analyses.

We enrolled 148 participants with metabolic syndrome and 206 controls, and their characteristics are summarized in Table 1.

The average age of participants with metabolic syndrome was significantly more than that of the controls, but there were not significantly more women or men in either group. BMI, blood pressure, fasting plasma glucose, total cholesterol, HDL cholesterol, and triglyceride were all significantly different between the metabolic syndrome and the control groups. Most characteristics in metabolic cases, with the exception of HDL-cholesterol, had higher average values than in the controls. However, LDL cholesterol levels were similar in both groups.

Serum sLOX-1 levels were higher in participants with metabolic syndrome [166.5 (136.5–182.1) pg/ mL] than in controls [63.3 (50.3-71.3) pg/mL], P < 0.001 (Figure 1A) with no significant differences between the sexes in either metabolic syndrome [161.05 ( 135.92-179.33) pg/mL in men vs 169.75 (136.42-191.25) pg/mL in women, P = 0.449] or control groups [58.14 (47.76-69.72) pg/mL in men vs 63.96 (51.10-72.21) pg/mL in women, P = 0.503)] (Figure 1B).

Figure 1

Correlations of sLOX-1 levels with classical metabolic risk factors and serum lipid profiles were analyzed and the results are shown in Table 2.

The circulating sLOX-1 levels were significantly correlated with age, BMI, blood pressure (BP), both systolic and diastolic. They were also correlated with FPG, total cholesterol, triglyceride, and HDL cholesterol, but not significantly correlated with sex or LDL cholesterol. Correlation between sLOX-1 and risk factors in Table 2 was analyzed by multiple linear regression and the results are shown in Table 3.

Results showed that age, SBP, BMI, FPG, and triglyceride were independently and significantly associated with sLOX-1 level.

Sensitivity and specificity of sLOX-1 were studied. At the cut-off value (114 pg/mL), sLOX-1 showed 99.0% vs 98.7% specificity and 100% vs 100% sensitivity in diagnosis of metabolic syndrome and control, respectively. The ROC curves presented a true positive fraction (sensitivity as Y axis) plotted against the false positive fraction (1 – specificity as X axis) for the diagnosis of metabolic syndrome. Area under the ROC curve was 0.998 (95%CI: 0.9961.001, P < 0.001), indicating that the level of sLOX-1 was highly sensitive and specific for metabolic syndrome.

The univariate and multivariate logistic regression analyses of several parameters for predictive index of metabolic syndrome are summarized in Table 4.

Univariate regression found that age older than 36 years, being overweight (BMI ≥25 kg/m²for women and ≥27 kg/m² for men), hypertension (SBP ≥130 mmHg, DBP ≥85 mmHg), hyperglycemia (FBG ≥110 mg/dL), hypertriglyceridemia (≥150 mg/dL), hypercholesterolemia (≥200 mg/dL), low HDL cholesterol (<40 mg/dL in men and <50 mg/dL in women), and high sLOX-1 level (>114 pg/mL) are significantly associated with metabolic syndrome.

Multivariate regression analysis found that being overweight, hyperglycemia, and hypertriglyceridemia produced an 82.3- (95%CI 10.7-631.9), 1.1- (95%CI 1.1-1.2), and 1.1- (95%CI 1.0-1.1)-fold higher risk of having metabolic syndrome, after adjusting for sex, age, SBP, DBP, BMI, and other variables (Table 4). HDL cholesterol level indicated a protective factor for metabolic syndrome of 0.96 (95%CI: 0.93-0.99).