Association of Human Adenovirus-36 With Dyslipidemia in Tehranian Children and Adolescent; TLGS

Background: The human adenovirus-36 (Adv36) has been associated with obesity and lipid disorders in some countries. The primary dyslipidemia related to obesity is characterized by increased TG, decreased HDL levels and abnormal LDL composition. Childhood obesity is a major public health problem in most developing countries.

Objectives: The aim of this study was to investigate the association between Adv36 and lipid disorders in Tehranian youth.

Patients and Methods: In a cross-section/observational study, anthropometric, blood pressure and biochemical measurements were examined in 54 youth, aged below 19 years, selected randomly from participants of Tehran Lipid and Glucose Study (TLGS) of Iran. Human Adv36 antibody of serum was determined using ELISA method.

Results: Of 54 youth, with a mean age of 14.78 ± 2.62 years, 85.2% were Adv36 seropositive (N = 46) and 14.8% were seronegative (N = 8). Subjects with Adv36 seropositive had higher mean of age, weight, TC, LDL-C, TG and SBP and lower level of HDL-C, waist, FBS and DBP. The unadjusted OR for elevated TG increased in participants who were Adv36 seropositive (OR 3.062, 95% CI: 0.344-27.293). Although the unadjusted OR for other lipid variables, such as elevated TC and LDL-C or decreased HDL-C, tended to increase in participants who were Adv36 seropositive (OR 1.028, 95% CI: 0.996 – 1.060; OR 1.057, 95% CI: 0.107 – 10.481; OR 1.125, 95% CI: 0.238 – 5.325, respectively), the results were not significant.

Conclusions: It seems that seropositive Adv36 has a strong association with lipid disorders, especially elevated TG level in Tehranian children and adolescent.

Primary dyslipidemia related to obesity is characterized by increased triglycerides (TG), decreased HDL levels and abnormal LDL composition. Much work has been performed to elucidate the pathogenesis of dyslipidemia in obesity ( 1 , 2 ). All of the components of dyslipidemia have been shown to be atherogenic. Increase in cardiovascular risk depends to a significant extent on changes in lipid profiles as observed in obesity ( 3 – 5 ). Various treatment protocols for dyslipidemia and coronary artery disease have been suggested. Combined therapy is recommended for treating dyslipidemia in high-risk patients for cardiovascular problems ( 6 ). Childhood obesity is a major public health problem in most developing countries. Most obese children remain obese during adulthood and this can lead to hypertension, dyslipidemia, type II diabetes, coronary heart disease and psychological problems. Although, obesity has been considered as a multifactorial disorder, the concept of a viral origin has been less studied in comparison with genetic and other behavioral causes. Studies revealed that obesity is associated with several pathogens, such as human adenovirus-36 (Adv36), Adv37 and SMAM-1 ( 7 , 8 ). Investigators showed that infection with a virus such as Adv36 enhanced adipose tissue and obesity ( 9 ). The mechanisms of increase in lipogenic enzymes of adipose tissue appear to be due to an effect of the E4orf1 gene of Ad-36 ( 10 ). Moreover, it has been revealed that Adv36 infection of some animal species, not human, resulted in obesity through increasing proliferation and differentiation of preadipocytes and lipid accumulation in mature adipocytes ( 10 – 14 ). The association between Adv36 and obesity in adults differs between most subsequent studies being somewhat inconsistent, but the findings in children consistently associate Adv36 infection with obesity and dyslipidemia. A study performed on American adult, using serology to determine previous exposure to Adv36, showed that 30% of obese individuals were seropositive compared to 11% of non-obese subjects ( 15 ). A recent uncontrolled study of obese children in Korea found Adv36-specific antibodies in 28.6% of the studied sample ( 16 ). In addition, other studies on Italian and US children and adolescent revealed the association between Adv36 antibody and obesity ( 17 , 18 ). Although these results are interesting regarding the ‘Infect-obesity’ concept, ethnic groups other than Americans should be tested before the concept can be generalized. In contrast, it was reported that Adv36 has a minor role in obesity in Danish population, as its prevalence is very low (5%) ( 19 ). Positivity of Adv36 antibody has also been associated with lower TG and total cholesterol (TC) in chickens, mice and nonhuman primates ( 20 ). Moreover, the association between Adv36 infection and lipid disorders was evident in a human adult sample with a mean age of 35 years. However, existence of such a relationship has yet to be examined in children and adolescent.

Dyslipidemia associated with obesity plays a major role in the development of atherosclerosis and cardiovascular disease (CVD) and to our knowledge there is no investigation assessing the association between Adv36 and lipid disorders in Tehranian youth. Therefore, we aimed to determine the association between antibody statuses of Adv36 with serum lipid profiles in Tehranian children and adolescent.

3.1. Study Population

In the current study, subjects aged below 19 years who lacked complete data for biochemical and anthropometric variables were excluded. This study was a short report of an ongoing project, in which a total of 348 subjects including 294 adults and 54 youths, were selected randomly from Tehran Lipid and Glucose Study (TLGS) participants of Iran. Sampling was used according to the literatures ( 18 , 21 ). Tehran Lipid and Glucose Study is an ongoing prospective population-based longitudinal cohort study, conducted to determine the risk factors for non-communicable diseases among a representative Tehran urban population ( 22 ). The design of TLGS includes two major components, a cross-sectional prevalence study of CVD and associated risk factors and a prospective 20-year follow-up, in several phases at about 3.6-year intervals; phase I 1999-2001, phase II 2002 – 2005 and phase III 2006 – 2008. In the present study, individuals were recruited from phase III of TLGS. Participants provided an informed consent and the study was approved by the Eighth institutional ethics committee (code 286ec88/12/11) of Research Institute for Endocrine Sciences, affiliated to Shahid Beheshti University of Medical Sciences, Tehran, Iran. Data on age, sex, demographic and medication usage for treatment of diabetes, hypertension and lipid disorders were collected, using a standardized questionnaire. Weight was measured and waist circumference (WC) was measured at the umbilical site using an outstretched tape meter to the nearest 0.1 cm. Systolic and diastolic blood pressures (SBP and DBP) were measured in the sitting position with a standard mercury sphygmomanometer on the left arm, after at least 10 minutes of rest. Blood samples were taken after 12 – 14 hours of overnight fasting for biochemical analysis, in a standard sitting position and then centrifuged during 45 minutes after collection. All the assays including fasting blood glucose (FBS), triglycerides (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) were performed on the day of sampling. FBS level was measured by glucose Oxidase method (Glucose kit; Pars Azmun, Tehran, Iran). Plasma TG and TC for the samples were determined by an enzymatic colorimetric method (TG and TC kit; Pars Azmun, Tehran, Iran). HDL-C and LDL-C samples were also determined by precipitation and an enzymatic colorimetric method (HDL-C and LDL-C kit; Pars Azmun, Tehran, Iran). Obesity was defined based on standardized percentile curves of BMI suggested for Iranian children and adolescents. Overweight/obese individuals were defined as ≥ 85th percentile of BMI for age and sex, while normal weight was determined as < 85th percentile of BMI for age and sex ( 23 ). The cutoff points for lipid disorders were modified from age-modified standards of the National Cholesterol Education Program (NCEP)-Adult Treatment Panel (ATP) III metabolic syndrome criteria ( 24 ).

3.2. Serum Antibody Assay

For serum preparation, the blood samples from antecubital vein of participants’ centrifuged 10 minutes at 3000 rpm. The presence of Adv36-specific neutralizing antibodies was assessed using specific ELISA kit (Human anti-AD36 ELISA kit, Cusabio Biotech Co. Ltd, Wuhan, China). The assay performed based on the kit insert.

3.3. Statistical Analysis

The Kolmogorov-Smirnov goodness-of-fit test, Skewness and QQ plot were used to assess the normal distribution of continuous data. Normally distributed continuous variables were reported as the Mean ± Standard Deviation (SD); whereas, categorical variables were summarized as frequencies and percentage. Logarithmic transformation was performed to normalize the distribution of HDL-C and TG. Baseline clinical and demographic covariates were summarized according to Adv36 status. The mean of variables were compared by a two-tailed Student’s t-test in infected versus uninfected individuals. The chi-square (x ² ) test was used to examine the distribution of categorical variables. Logistic and linear regression analysis was conducted to determine the odds ratio (OR) for lipid disorders according to the presence of Adv36 antibody. All statistical analyses were performed by SPSS software (version 20.0; SPSS, Chicago, IL, USA). Probability values < 0.05 were considered statistically significant.

Of 54 children and adolescent with a mean age of 14.78 ± 2.62 years, 85.2% were Adv36 seropositive (N = 46) and 14.8% were seronegative (N = 8). The demographic and clinical features of study samples are presented in Table 1 . Adv36 seropositive subjects had higher mean of weight, waist circumference, TC, LDL-C, TG, DBP and SBP and lower HDL-C levels than negative group, from that the differences were significant for TC, LDL-C, TG and SBP the (P < 0.05). The mean of BMI was not different significantly between the two studied groups. The results of logistic and linear regression analysis are shown in Table 2 . The unadjusted OR for the elevated TG increased in participants who were Adv36 seropositive (OR 3.062, 95% CI: 0.344-27.293; P = 0.316). This trend did not so much differ after adjustment for age and sex (OR 2.830, 95% CI: 0.310-25.822; P = 0.356). Although the unadjusted OR for other lipid variables such as elevated TC and LDL-C or decreased HDL-C, tended to increase in participants who were Adv36 seropositive (OR 1.028, 95% CI: 0.996-1.060; P = 0.075 for TC; OR 1.057, 95% CI: 0.107-10.481; P = 0.962 for LDL-C; OR 1.125, 95% CI: 0.238-5.325; P = 0.882 for HDL-C), the results were not statistically significant.

Table 1. Characteristics of Adv36 Positive and Negative Participants a

Table 2. OR and 95% CI for Adv36 Antibody Positivity According to the Presence of Lipid Disorders a, b

In this study on 54 individuals of Tehranian children and adolescent, we observed a strong association between Adv36 seropositive status and lipid disorders. Participants who were Adv36 seropositive had higher TG, TC, LDL-C and SBP and lower HDL-C levels than those who were Adv36 seronegative. These results are confirmed by a study performed in Korean population ( 21 ). These results suggested that the Adv36 antibody might be an independent risk factor for elevated TG, TC and LDL-C. As far as we know, this report is the first to show a strong association between Adv36 seropositive status and lipid disorders in Tehranian participants, especially for children and adolescent. Several reports have shown that Adv36 infection directly modifies adiposity factors in animals ( 9 , 13 , 25 ). Furthermore, the investigators of a twin study hypothesized that Adv36 infection also increases adiposity in human ( 26 ). Our study showed an association between Adv36 seropositivity and high TG, TC, LDL-C and SBP and low HDL-C levels, which were elevated in Adv36 seropositive youth. Several factors such as differences in race, age and ethnic-specific lifestyles and diet may explain the disparity between our results and those of the other reports. Infection with Borna disease virus, Canine distemper virus and Rous-associated virus type 7 was reported to induce obesity in rats, mice and chickens, respectively, all of which were associated with increased TG level ( 13 , 27 , 28 ). Furthermore, several reports have shown that infection with human immune deficiency virus is associated with increased TG ( 29 , 30 ). We also conducted multiple logistic and linear regression analysis to determine whether Adv36 is an independent risk factor for dyslipidemia in Tehranian children and adolescent. Our data showed that unadjusted OR for Adv36 seropositivity was elevated in individuals. Several reports have shown that Adv36 infection directly modifies adiposity agents in animals ( 9 , 13 , 25 , 27 ). Furthermore, the investigators of an elegant twin study hypothesized that Adv36 infection increases adiposity in humans ( 15 ). Our study provided further evidence for the association between dyslipidemia and Adv36 antibody for the first time in Tehran. As far as we know, this was the first study to show that Adv36 infection is strongly associated with lipid disorders in Tehranian children and adolescent. However, our results are subject to some limitations. First, the cross-sectional study design is limited to eliminate casual relationships between Adv36 and lipid profiles. Another is that because of limitation in outlay and expenditure to measure the antibody against Adv36, we used a relatively small and limited sample consisting of youth. Moreover, a large-scale study incorporating various ethnic groups and age groups should be conducted to evaluate the association between Adv36 infection and obesity or related disorders.

We would like to acknowledge Miss Hoda Ghadaksaz and Miss Laleh Hoghoughi for samples’ collection and doing the ELISA assay.