Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2021 Oct 15;16(10):e0258617. doi: 10.1371/journal.pone.0258617

Sex differences in the association of vitamin D and metabolic risk factors with carotid intima-media thickness in obese adolescents

Indah K Murni 1,2,*, Dian C Sulistyoningrum 3, Danijela Gasevic 4, Rina Susilowati 5, Madarina Julia 1
Editor: Raffaella Buzzetti6
PMCID: PMC8519449  PMID: 34653200

Abstract

Background

It has been shown that vitamin D is associated with obesity and the development of atherosclerosis. Less is known about this association among adolescents with obesity.

Objectives

To determine the association of vitamin D level and metabolic risk factors with carotid intima-media thickness (CIMT) among obese adolescents.

Methods

We conducted a cross-sectional study among obese children aged 15 to 17 years in Yogyakarta, Indonesia. The association of vitamin D and other metabolic risk factors (triglyceride, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), and insulin resistance using homeostasis model assessment of insulin resistance (HOMA-IR)) with CIMT was explored by multivariable linear regression models.

Results

Out of 156 obese adolescents, 55.8% were boys. Compared to girls, boys had higher BMI z-score, waist circumference, and HDL-cholesterol. After adjustment for age, sex and second-hand smoke exposure, high HOMA-IR, total cholesterol, LDL-cholesterol and triglyceride levels were associated with higher odds of elevated CIMT. In analyses stratified by sex, a similar trend was observed in boys, while none of the risk factors were associated with CIMT in girls. We observed no association between vitamin D and CIMT.

Conclusions

Hyperinsulinemia, higher total cholesterol and LDL cholesterol were associated with greater odds of elevated CIMT among obese adolescent boys.

Introduction

Atherosclerosis is a one of the important causes of cardiovascular and cerebrovascular diseases which lead to significant morbidity and mortality [1]. Subclinical atherosclerosis is an early indicator of atherosclerotic burden [2], and carotid intima-media thickness (CIMT) is regarded as a reliable marker of subclinical atherosclerosis since increased CIMT can reflect an increase in arterial wall thickness [3] including in children and adolescents [4]. Increased CIMT has been associated with obesity in children, familial hypercholesterolemia, type 1 diabetes, hypertension [5] and vitamin D deficiency [4].

Vitamin D is a steroid hormone with both endocrine and autocrine functions [6]. The endocrine function of vitamin D is mainly on the maintenance of calcium homeostasis and bone metabolism [7]. One of the autocrine functions of vitamin D is the modulation of inflammatory pathways which plays a role in cardiovascular diseases [8]. Vitamin D deficiency has been associated with the development of atherosclerotic and abnormality of arterial wall thickness mostly in adults [3, 9]. Vitamin D may play an important role in the endothelial or smooth muscle vascular cells and can also be involved in immune or inflammatory modulation [10, 11]. Therefore, vitamin D deficiency may contribute to an imbalance of vascular homeostasis, decreased arterial compliance, and the development of atherosclerosis [11]. Sufficient level of vitamin D in adults leads to a reduction in circulating inflammatory and endothelial function biomarkers; therefore, vitamin D might have a potential role as an anti-inflammatory therapy for the prevention and the treatment of cardiovascular disease [9]. Deficiency of vitamin D might be common in obese populations because of reduced sun exposure due to increased sedentary activities and sequestration of vitamin D in lipocytes [12]. There were few studies reporting the association between vitamin D deficiency with increased CIMT in children and adolescents with conflicting results [4, 13–15].

Studies evaluating the relationship of vitamin D deficiency and increased CIMT in obese adolescents are few and most studies were conducted in high-income countries among Caucasian populations [13–17]. Given limited data from low- and middle-income and tropical countries, studies are needed to better identify the association of vitamin D level and cardiovascular risk on vascular thickness among obese adolescents. This study aimed to determine the relationship of vitamin D status and other metabolic risk factors with subclinical atherosclerosis among obese adolescents in Indonesia (a low-to-middle income country with naturally abundant sun exposure). We were particularly interested in looking at CIMT as predicted by vitamin D status independent of other predictors of CIMT.

Methods

Study design and population

We conducted a cross-sectional study among adolescents in Yogyakarta, Indonesia. The recruitment process was done by screening for adolescents with obesity in seven public and three private high schools in Yogyakarta, a city in the Southern part of Java, Indonesia. The participants were recruited at their schools and we screened 4,268 students from 10 schools in Yogyakarta using three body mass index (BMI) reference cut-off points: World Health Organization (WHO) [18], Centre for Disease Control and Prevention (CDC) [19], and International Obesity Task Force (IOTF) [20]. BMI was converted into BMI z-scores based on WHO Growth Reference 2007 using WHO AnthroPlus (https://www.who.int/growthref/tools/en/). The screening was done from January to February 2016. Obesity was considered when fulfilling all of the three obesity criteria [18–20]. Obese adolescents who agreed to participate in the next step of the study, i.e. assessment of their metabolic risk, were included in this study. The blood collection was taken at their high schools by a trained laboratory technician, while the assessment of carotid intima media thickness was performed at the Dr Sardjito Hospital, Yogyakarta. These data collections were done from March to October 2016.

Inclusion criteria were obese children aged 15 to less than 18 years who agreed to participate in this study. We excluded children with diabetes mellitus, renal disease, cardiovascular disease, any history of any systemic disease or history of current steroid use. Approval for the study was obtained from the Ethics Committee of the Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia (KE/FK/333/EC/2016). Written informed consent was obtained from all parents/guardians included in the study.

Data collection

All eligible children underwent history taking, physical examination, and blood collection. We collected demographic information as well as information on smoking exposures and family history of hypertension. We measured body weight, height, and waist circumference. The children’s weight was measured using a portable weighing scale (CAMRY, EB9003) while they were in light clothing without shoes or slippers. The weight was recorded in kilograms (kg) to the nearest 0.1 kg. We measured height using a portable stadiometer or microtoise (GEA), and height was recorded in centimeters (cm) to the nearest 0.1 cm.

Waist circumference was measured using standardised procedures by placing a tape midway of the hipbone and the bottom of ribs and wrapping it around the child’s waist. Abdominal obesity was defined as waist to height ratio ≥ 0.5 [21].

Blood pressure was reported as the average of three measurements collected after a 10-minute rest. Elevated blood pressure, including hypertension, was defined according to the Clinical Practice Guidelines for Screening and Management of High Blood Pressure in Children and Adolescents proposed by The American Academy of Pediatrics 2017. For adolescents aged ≥13 years, elevated blood pressure was systolic blood pressure of ≥120 mmHg, irrespective of diastolic blood pressure [22].

A total of 10 ml blood was collected to measure serum levels of triglyceride, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting blood glucose, insulin, and glycated hemoglobin (HbA1c). Fasting plasma lipid profile was measured using enzymatic assays. Increased risk of diabetes and insulin resistance were assessed using HBA1c, fasting plasma glucose, fasting insulin and homeostasis model assessment of insulin resistance (HOMA-IR). Fasting plasma insulin was measured using immunoassay, while the fasting plasma glucose was measured using the hexokinase method. HOMA-IR was calculated from fasting plasma glucose and insulin using the specified formula: (fasting insulin (microU/L) x fasting glucose (nmol/L)/22.5) [23].

For impaired glucose metabolism, the criteria defined by the American Diabetes Association for increased risk of diabetes was fasting glucose ≥100 mg/dL and HbA1c ≥5.7% [24]. Insulin resistance was defined when HOMA-IR was >2.5 [24]. High insulin level was defined as insulin level above 15.7 microU/mL [25].

To define dyslipidemia, we used pediatric-specific cut-off values identified by the National Cholesterol Education Program (NCEP) Expert Panel on Cholesterol Levels in Children for: high triglyceride ≥130 mg/dL, high total cholesterol ≥200 mg/dL, LDL cholesterol ≥130 mg/dL, HDL cholesterol <40 mg/dL [26].

Level of vitamin D was measured using enzyme-linked immunoabsorbent assay or ELISA (DRG® 25-Hydroxyvitamin D Total ELISA, EIA 5396), which had been validated in pediatric population [27]. Vitamin D status was defined as deficiency of serum vitamin D level at < 20 ng/mL, insufficiency of serum vitamin D level 20–30 ng/mL, and sufficient for serum vitamin D level at >30 ng/mL [4, 8].

Carotid intima-media thickness (CIMT) was measured using common carotid artery B-mode ultrasound [2]. The carotid arteries were imaged using a standard echocardiography machine of Phillips HD 15 with vascular probe L12.3. CIMT was measured on the posterior (far) wall of the left carotid artery at end diastolic phase. At least three measurements were taken at approximately 10 mm proximal to the bifurcation to derive mean CIMT. A trained technician who measured the carotid arteries was blinded to the subject’s clinical information. Abnormal CIMT was determined if the value was ≥ 95th percentile for age, sex, and height. Elevated CIMT was defined when CIMT measurement ≥ 0.047 mm in girls and ≥ 0.049 mm in boys [28].

Statistical analysis

Data analyses were performed using STATA version 12.1, StataCorp LP, Texas. Continuous data are presented as mean and standard deviation (SD) or median and quartiles 1 (Q1) and Q3, for normally distributed and skewed data, respectively. Categorical variables are presented as counts and percentages. Normality of variables was checked using Kolmogorov-Smirnov tests.

Normally distributed data are compared using independent sample t-test, while skewed data are compared using independent-samples Mann-Whitney U test. Categorical data are presented as percentages and compared using chi-square tests. The strength of correlation between vitamin D and metabolic risk factors with carotid intima-media thickness between boy and girl obese adolescents were analyzed using Pearson correlation.

The dependent variable was CIMT as a marker of early vascular impairment. The independent variables were vitamin D and other metabolic risk factors including waist-to-hip ratio (marker of abdominal obesity), blood glucose, HbA1c (impaired glucose metabolism), fasting insulin, HOMA-IR (insulin resistance), LDL and HDL cholesterol, triglyceride (impaired lipid metabolism), blood pressure, smoking and family history of hypertension.

The association of vitamin D and other metabolic risk factors with CIMT was explored using logistic regression and presented as odds ratio (OR) with 95% confidence interval (CI). Each factor was tested in a separate regression model. Models that were significantly associated with elevated CIMT, were re-tested with adjustment for age, sex and secondary smoke exposures. All analyses were performed on a sample as a whole and also stratified by sex based on biological differences in body fat accumulation and potentially vitamin D levels between boys and girls.

Results

We screened 4,268 students from seven public and three private high schools in Yogyakarta, Indonesia and identified 298 (7%) adolescents classified as obese based on all three obesity criteria of World Health Organization (WHO) [18], Centre for Disease Control and Prevention (CDC) [19], and International Obesity Task Force (IOTF) [20]. Of those, 229 (76.8%) obese adolescents agreed to participate in the next step of the study, i.e. assessment of their metabolic risk. The results of this assessment have been reported elsewhere [29].

This study included 156 (out of 229) obese adolescents who had data on CIMT. There were no differences in metabolic risk parameters between adolescents who had their CIMT measured versus those who had not (S1 Appendix). The exceptions were triglyceride levels that were on average higher among adolescents with data missing on CIMT. We considered that the sample of this study could be considered representative of a larger population.

Vitamin D deficiency (vitamin D at < 20 ng/mL) was observed in 30.1% of children, while 13.6% had vitamin D insufficiency (vitamin D level 20–30 ng/mL); and the rest of children (56.4%) had sufficient vitamin D level (vitamin D level at >30 ng/mL) (Fig 1).

Fig 1. Prevalence of increased carotid intima media thickness across vitamin D status among obese adolescents.

Fig 1

Open in a new tab

Compared to girls, boys had significantly higher BMI z-score, waist circumference, and HDL cholesterol (Table 1). We found no correlation between vitamin D and CIMT, but LDL and total cholesterols were positively correlated with carotid intima media thickness among obese adolescent boys (Table 2). Boys with elevated CIMT showed increased values of LDL cholesterol (Table 3). Table 4 demonstrated that, overall, elevated HOMA-IR, total cholesterol, LDL-cholesterol and triglyceride levels were associated with greater odds of elevated CIMT. The associations persisted after adjustment for age, sex and second-hand smoke exposure. However, after the data were analyzed within each sex, similar results were only observed in boys. None of the risk factors were associated with CIMT in girls.

Table 1. Characteristics of between obese adolescent boys and girls from Yogyakarta, Indonesia.

Variables All subjects Boys Girls p value
n = 156 (%) n = 87 (%) n = 69 (%)
Age, years 16.4 (0.7) 16.3(0.7) 16.5(0.6) 0.030
Weight, kg 85.2 (78.0;95.7) 91.7 (84.3;101.0) 78.1 (73.0;84.6) <0.001
Height, cm 162.7 (9.2) 168.6(7.0) 155.3(5.5) <0.001
BMI z-scores 2.54 (2.30;2.90) 2.62 (2.34;3.02) 2.48 (2.26;2.74) 0.004
Waist circumference, cm 93.3 (86.3;98.6) 96.6 (92.3;104.5) 86.1 (83.2;92.3) <0.001
Waist-to-Height Ratio 0.57 (0.54;0.60) 0.58 (0.54;0.62) 0.56 (0.53;0.59) 0.007
Systolic blood pressure, mmHg 115.0 (106.0;124.0) 119.0 (110.0;127.0) 111.0 (102.0;120.0) <0.001
Diastolic blood pressure, mmHg 74.0 (69.0;81.0) 73.0 (68.0;81.0) 75.0 (70.0;82.0) 0.143
High waist-to-hip ratio, ≥ 0.5a 151 (96.8) 87 (100) 64 (92.8) 0.002 *
High waist-to-hip ratio, ≥ 0.6 a 39 (25.0) 28 (32.2) 11(15.9) 0.020
Vitamin D ≤30 ng/mL b 68 (43.6) 43 (49.4) 25 (36.2) 0.099
Vitamin D ≤20 ng/mL b 47 (30.1) 31(35.8) 16 (23.2) 0.092
Elevated fasting insulin, ≥ 15.7 mg/dL 134 (86.5) 78 (89.7) 56 (82.4) 0.187
Elevated fasting blood glucose, ≥ 100 mg/dL 6 (3.8) 4 (4.6) 2 (2.9) 0.584*
HOMA-IR, >2.5 146 (96.2) 83 (95.4) 63 (92.6) 0.467*
High HbA1C, ≥ 5.7% 6 (3.8) 3 (3.4) 3 (4.3) 0.772*
High total cholesterol, ≥ 200 mg/dL 32 (20.5) 14 (16.1) 18 (26.1) 0.125
High LDL cholesterol, ≥ 130 mg/dL 50 (32.1) 28 (32.2) 22 (31.9) 0.968
Low HDL cholesterol, < 40 mg/dL 41 (26.3) 29 (33.3) 12 (17.4) 0.025
High triglyceride, ≥ 130 mg/dL 58 (37.2) 38 (43.7) 20 (29.0) 0.062
Active smoking 10 (6.4) 10 (11.5) 0 (0.0) 0.002 *
Second hand smoke exposure 105 (67.3) 62 (71.3) 43 (62.3) 0.237
Family history of hypertension 47 (30.1) 28 (32.2) 19 (27.5) 0.530
Elevated CIMT (≥0.47 mm in girls, ≥ 0.49 mm in boys) 23 (14.7) 15 (17.2) 8 (11.6) 0.323
Open in a new tab

Normally distributed and skewed continuous data are presented as mean (standard deviation or SD) and median (Quartile 1 or Q1;Q3), respectively. Normally distributed data are compared using independent sample t-test, while skewed data are compared using independent-samples Mann-Whitney U test. Categorical data are presented as n (%) and compared using chi-square tests unless otherwise indicated. P values report the significance comparing boys and girls.

* Fischer Exact Test.

a Different cut off was used: high waist-to-hip ratio ≥ 0.5 or ≥ 0.6.

b Different cut off was used: Vitamin D level ≤30 ng/mL or ≤20 ng/mL.

BMI = body mass index; HOMA-IR = homeostatic model assessment of insulin resistance; HbA1C = glycated hemoglobin, LDL = low density lipoprotein; HDL = high density lipoprotein; CIMT = carotid intima media thickness.

Table 2. Correlations between of vitamin D levels and other metabolic risk factors to predict carotid intima-media thickness in obese adolescent boys and girls.

Boys (n = 87) Girls (n = 69)
ρ p ρ p
Age, years -0.027 0.801 0.044 0.717
Waist-to-hip ratio 0.119 0.273 -0.144 0.237
Systolic blood pressure, mmHg 0.078 0.472 0.094 0.444
Diastolic blood pressure, mmHg -0.032 0.767 0.104 0.396
Vitamin D, ng/mL 0.077 0.481 0.163 0.182
Fasting insulin, mg/dL 0.104 0.339 -0.127 0.303
Fasting blood glucose, mg/dL -0.139 0.198 0.082 0.501
HOMA-IR 0.088 0.420 -0.125 0.309
HbA1C, g/dL -0.015 0.889 0.114 0.349
Total cholesterol, mg/dL 0.316 0.003 -0.029 0.814
LDL cholesterol, mg/dL 0.337 0.001 -0.012 0.920
HDL cholesterol, mg/dL -0.085 0.433 -0.132 0.279
Triglyceride, mg/dL 0.152 0.160 0.105 0.392
Open in a new tab

LDL = low density lipoprotein; HDL = high density lipoprotein; HOMA-IR = homeostatic model assessment of insulin resistance; HbA1C = glycated hemoglobin.

Table 3. Differences of vitamin D levels and other metabolic risk factors in obese adolescent boys and girls with elevated carotid intima-media thickness and not elevated carotid intima-media thickness.

Variables Boys (n = 87) Girls (n = 69)
Elevated CIMT n = 15 Not Elevated p value Elevated CIMT Not Elevated p value
n = 72 n = 8 n = 61
Age, years 16.15 (0.81) 16.32 (0.67) 0.394 16.56 (0.47) 16.52 (0.61) 0.849
Systolic blood pressure, mmHg* 116.0 (107.0;124.0) 119.5 (108.5;126.0) 0.749 115.5 (107.8;119.0) 110.0 (102.5;119.0) 0.343
Diastolic blood pressure, mmHg* 71.0 (69.0;80.0) 72.0 (67.3;80.0) 0.951 79.5 (72.0;84.5) 74.0 (70.0;79.0) 0.212
BMI, kg/m2 34.08 (4.51) 32.67 (3.70) 0.199 33.53 (3.86) 32.64 (2.91) 0.435
BMI z-scores 2.86 (0.49) 2.67 (0.43) 0.136 2.63 (0.46) 2.53 (0.35) 0.474
Waist circumference, cm 101.7 (10.6) 97.9 (9.4) 0.163 87.0 (7.4) 86.7 (7.1) 0.908
Waist-to-Height Ratio* 0.59 (0.55;0.64) 0.57 (0.54;0.61) 0.096 0.55 (0.52;0.60) 0.55 (0.52;0.58) 0.779
Vitamin D, ng/mL* 21.9 (16.0;51.3) 30.9 (17.3;49.1) 0.835 45.4 (32.4;57.0) 36.9 (21.1;53.6) 0.358
Insulin, μIU/mL* 35.6 (13.9;64.2) 35.9 (24.0;55.2) 0.991 24.7 (16.0;48.2) 30.1 (19.5; 44.4) 0.775
Fasting Plasma Glucose, mg/dL 84.80 (6.93) 86.85 (6.63) 0.283 89.38 (11.90) 86.38 (15.74) 0.606
HOMA-IR* 7.0 (2.8–11.4) 6.6 (4.4;10.1) 0.937 4.9 (3.0; 9.4) 5.5 (3.9;7.9) 0.805
HbA1C, % 5.26 (0.26) 5.20 (0.27) 0.448 5.25 (0.39) 5.16 (0.98) 0.801
Cholesterol, mg/dL 187.9 (24.9) 172.6 (30.3) 0.071 191.6 (47.0) 174.8 (31.9) 0.190
LDL cholesterol, mg/dL 131.9 (21.3) 116.7 (27.9) 0.050 133.0 (42.0) 116.5 (29.5) 0.160
HDL cholesterol, mg/dL 43.13 (7.43) 42.76 (8.12) 0.871 41.75 (11.94) 48.43 (8.86) 0.059
Triglyceride, mg/dL * 141.0 (95.0;173.0) 118.5 (94.0;168.0) 0.609 133.0 (83.5; 203.7) 95.0 (73.5;133.5) 0.097
Open in a new tab

Normally distributed and skewed continuous data are presented as mean (standard deviation or SD) and median (Quartile 1 or Q1;Q3), respectively. Normally distributed continuous data are compared using independent sample t-test, while skewed data are compared using independent-samples Mann-Whitney U test. Categorical data are presented as n (%) and compared using chi-square tests unless otherwise indicated. BMI = body mass index; HOMA-IR = homeostatic model assessment of insulin resistance; HbA1C = glycated hemoglobin, LDL = low density lipoprotein; HDL = high density lipoprotein; CIMT = carotid intima media thickness.

Table 4. The association between vitamin D levels and other metabolic risk factors to predict carotid intima-media thickness among obese adolescent boys and girls.

All subjects (n = 156) Boys (n = 87) Girls (n = 69)
OR (95%CI) P OR (95%CI) p OR (95%CI) p
Age, years 0.76 (0.39–1.50) 0.430 0.69 (0.30–1.60) 0.390 1.13 (.32–3.99) 0.846
High waist-to-hip ratio, ≥0.5 a na Na na na na na
High waist-to-hip ratio, ≥0.6 a 2.21 (0.87–5.60) 0.096 2.13 (0.68–6.61) 0.193 1.93 (0.34–11.08) 0.463
Elevated blood pressure 1.03 (0.42–2.55) 0.948 1.42 (0.46–4.40) 0.545 0.70 (0.15–3.23) 0.645
Vitamin D ≤30 ng/mL 1.01 (0.41–2.46) 0.991 0.83 (0.27–2.52) 0.740 1.82 (0.34–9.76) 0.487
Vitamin D ≤20 ng/mL 1.66 (0.58–4.78) 0.346 1.65 (0.48–5.70) 0.429 2.28 (0.26–20.09) 0.457
Elevated fasting insulin, ≥ 15.7 mg/dL 2.75 (0.94–8.06) 0.065 4.87 (1.13–21.01) 0.034 d 1.67 (0.29–9.48) 0.565
Elevated fasting blood glucose, ≥ 100 mg/dL 0.86 (0.10–7.71) 0.859 na na 0.12 (0.007–2.08) 0.144
HOMA-IR, >2.5 5.35 (1.32–21.69) 0.019 c 17.75 (1.70–185.1) 0.016 d 2.00 (0.20–20.51) 0.559
High HbA1C, ≥ 5.7% 3.07 (0.53–17.83) 0.211 2.50 (0.21–29.50) 0.467 4.21 (0.34–52.65) 0.264
High total cholesterol, ≥ 200 mg/dL 3.88 (1.51–9.96) 0.005 c 5.33 (1.50–18.94) 0.010 d 3.36 (0.74–15.18) 0.116
High LDL cholesterol, ≥ 130 mg/dL 3.37 (1.36–8.36) 0.009 c 4.48 (1.31–13.33) 0.015 d 2.39 (0.54–10.61) 0.252
Low HDL cholesterol, < 40 mg/dL 1.62 (0.63–4.15) 0.319 1.00 (0.72–6.92) 1.00 3.47 (0.70–17.11) 0.127
High triglyceride, ≥ 130 mg/dL 2.54 (1.03–6.25) 0.042 c 2.22 (0.72–6.92) 0.168 2.81 (0.63–12.59) 0.176
Active smoking na Na na na na na
Second hand smoke exposure 1.45 (0.53–3.93) 0.466 1.76 (0.45–6.86) 0.416 1.01 (0.22–4.62) 0.991
Family history of hypertension 1.61 (0.64–4.03) 0.311 1.52 (0.48–4.78) 0.478 1.69 (0.36–7.88) 0.506
Open in a new tab

Abnormal conditions were given code = 1, normal = 0; LDL = low density lipoprotein; HDL = high density lipoprotein; HOMA-IR = homeostatic model assessment of insulin resistance; HbA1C = glycated hemoglobin; na = not applicable.

a Different cut off was used: high waist-to-hip ratio ≥ 0.5 or ≥ 0.6.

b Different cut off was used: Vitamin D level ≤30 ng/mL or ≤20 ng/mL, *≥0.47 mm in girls, ≥ 0.49 mm in boys.

c The association remained significant after controlling for age, gender and second hand smoke exposure.

d The association remained significant after controlling for age and second hand smoke exposure.

The dependent variable was CIMT as a marker of early vascular impairment.

The independent variables were vitamin D and other metabolic risk factors including waist-to-hip ratio (marker of abdominal obesity), blood glucose, HbA1c (impaired glucose metabolism), fasting insulin, HOMA-IR (insulin resistance), LDL and HDL cholesterol, triglyceride (impaired lipid metabolism), blood pressure, smoking and family history of hypertension.

Discussion

This study explored the relationship of vitamin D and other metabolic risk factors with CIMT in 156 obese adolescents in Indonesia. The results of the study indicate a positive association of hyperinsulinemia, hypercholesterolemia, and LDL hypercholesterolemia with CIMT among boys. No association was observed between vitamin D and CIMT among obese adolescents.

In our study, only about half of the Indonesian obese adolescents had normal vitamin D level, and vitamin D deficiency was observed in 30.1% of the adolescents. This is lower than the prevalence of vitamin D deficiency noted for adolescents aged 10–18 years from other countries (49% [30], 75% [31]); however, there are other studies that reported a lower prevalence of vitamin D deficiency than that observed in our study (4.5% [14], 20% [15]). We also observed the higher prevalence of vitamin D deficiency in boys compared to girls. In another study from Indonesia where researchers recruited 120 children between ages 7 and 12 years, there were 15% of adolescents with deficient vitamin D levels [32]. They observed that 75.8% of adolescents were with insufficient vitamin D levels, which was higher than that observed among our study participants (13.6%). They also observed the higher prevalence of vitamin D insufficiency in girls compared to boys, which is in contrast to the findings of our study [32]. This discrepancy in findings may be due to differences in study sample, whereby most of recruited subjects were girls (62.5%) in the other Indonesian study, while in our study the proportion of girls was 44.2%. We also only recruited obese adolescents, and mean BMI z-scores in boys (2.7) were higher compared to girls (2.5). Vitamin D has been considered to be lower in people with obesity because it is sequestered in their lipocytes [12].

We observed no association between vitamin D and CIMT in obese adolescents. This is consistent with the results of previously published studies in adolescents from Italy [14], and the United States [15, 16]. However, our results are in contrast to the studies from Turkey [4] and the United States [13], which noted a positive association between vitamin D deficiency and CIMT. We extended the results of previous studies by reporting a positive association of LDL hypercholesterolemia, total hypercholesterolemia, hypertriglyceridemia and hyperinsulinemia with CIMT among obese adolescent boys. It is important to note that atherosclerosis can start in childhood showing lipid accumulation in the arterial intima [33]. Initially, children with atherosclerosis have at least some degrees of aortic fatty streaks [34, 35] and atherosclerotic plaques can be found in the coronary arteries during adolescence [35].

We observed no association between vitamin D and subclinical atherosclerosis in obese adolescents. It is possible that the lack of observing the association is due to vitamin D only serving as a marker for sunlight exposure [17, 36]. It is also likely that vascular changes may occur only after a significant period of vitamin D deficiency exposure, and therefore, the vascular effects may manifest later in children with chronic vitamin D deficiency [15]. Since we only measured vitamin D serum once, this may not reflect a long-term period of vitamin D deficiency. Therefore, our study population may have lacked time to develop atherosclerosis from vitamin D deficiency. In addition, a simultaneous measurement of inflammatory markers, parathyroid hormone, and estrogen with vitamin D level may strengthen the studies on the relationship of vitamin D and subclinical atherosclerosis [11, 37].

One study reported associations of vitamin D deficiency with adverse cardiovascular risk factors in children and adolescents since receptors of vitamin D can be found in vascular smooth muscles, endothelium, and cardiomyocytes [35]. Vitamin D deficiency contributes to the development of cardiovascular disease by promoting vascular stiffness and calcification, which lead to atherosclerosis [38]. Vitamin D deficiency is also associated with hypertension because of the renin-angiotensin-aldosterone system activation and endothelial system dysfunction [11, 39]. These can lead to the development of plaque as a degenerative vascular process that might result in myocardial infarction or stroke; and these processes start at a younger age [40].

The role of vitamin D deficiency in the development of atherosclerosis is also mediated by systemic and vascular inflammation [8, 11]. These include increased levels of inflammatory cytokines, such as C- reactive protein (CRP), tumor necrosis factor-α (TNF-α), and interleukin-6, and low levels of interleukin-10 [11]. The role of vitamin D on vascular smooth muscle cells is also modulated by parathyroid hormones and estrogen [37]. Vitamin D and estrogen have already proven to help prevent Metabolic Syndrome and cardiovascular diseases in postmenopausal women [37].

We demonstrated the association of LDL hypercholesterolemia and hyperinsulinemia with CIMT in obese adolescent boys. This message could inform policy makers to formulate an effective prevention strategy for the development of cardiovascular diseases in obese adolescents. Vitamin D deficiency, obesity and its comorbidities and particularly high levels of insulin, HOMA-IR, LDL cholesterol, and total cholesterol should be prevented among adolescents, and boys especially, to avoid the development of cardiovascular disease.

The strength of our study is that it is among the first to evaluate the association of vitamin D and metabolic disease risk factors with vascular thickness in obese adolescents living in a country where sun exposure is abundant. However, the study is limited by its cross-sectional design and small sample size, and as a result, we may have not had enough power to detect the association between vitamin D and CIMT. In addition, since this study was only performed in the city of Yogyakarta, the results could not be generalized to other obese adolescents in Indonesia, or other low- and middle-income country settings with abundant sun exposure. More robust studies with larger sample sizes and using a longitudinal design are needed to further explore the association between vitamin D and subclinical atherosclerosis among obese adolescents living in low- and middle-income countries with abundant sun exposure.

Conclusions

This study explored the relationship of vitamin D and metabolic disease risk factors with CIMT in obese adolescents in Indonesia. The results indicate a positive association of insulin resistance and dyslipidemia with CIMT among obese adolescent boys, while no association was observed between vitamin D and subclinical atherosclerosis in obese adolescents.

Supporting information

S1 Appendix. Characteristics of obese adolescents who had and had not CIMT measured.

(DOCX)

Click here for additional data file. (14.7KB, docx)
S1 Fig. Prevalence of increased carotid intima media thickness across vitamin D status among obese adolescents.

(TIFF)

Click here for additional data file. (217.8KB, tiff)
S1 Data. Dataset of vitamin D and CIMT.

(XLSX)

Click here for additional data file. (134.3KB, xlsx)

Acknowledgments

We would like to thank the Indonesian Pediatric Association Committee and Frisian Flag Indonesia (subsidiary of Friesland Campina) for funding this project. We also gratefully acknowledge Erik C Hookom for providing the editorial assistance.

Data Availability

All relevant data are within the paper and its Supporting Information file.

Funding Statement

we declare that the study was funded by the Indonesian Pediatric Society and Frisian Flag Indonesia (355/Legal/FFI/XII/2014). There was no additional external funding received for this study.

References

  • 1.World Health Organization. Cardiovascular diseases (CVDs) Fact sheet, 2017. http://www.who.int/mediacentre/factsheets/fs317/en/. Accessed by 8 Dec 2019.
  • 2.Holewijn S, den Heijer M, Stalenhoef AF, de Graaf J. Non-invasive measurements of atherosclerosis (NIMA): current evidence and future perspectives. Neth J Med 2010; 68: 388–99. [PubMed] [Google Scholar]
  • 3.Chen FH, Liu T, Xu L, Zhang L, Zhou XB. Association of Serum Vitamin D Level and Carotid Atherosclerosis: A Systematic Review and Meta-analysis. J Ultrasound Med 2018;37:1293–303 doi: 10.1002/jum.14494 [DOI] [PubMed] [Google Scholar]
  • 4.Atabek ME, Eklioglu BS, Akyürek N, Alp H. Association between vitamin D level and cardiovascular risk in obese children and adolescents. J Pediatr Endocr Met. 2014;27:661–6. doi: 10.1515/jpem-2013-0379 [DOI] [PubMed] [Google Scholar]
  • 5.Gilardini L, Pasqualinotto L, Di Matteo S, Caffetto K, Croci M, et al. Factors associated with early atherosclerosis and arterial calcifications in young subjects with a benign phenotype of obesity. Obesity 2011;19:1684–89. doi: 10.1038/oby.2010.342 [DOI] [PubMed] [Google Scholar]
  • 6.Morris HA, Anderson PH. Autocrine and paracrine action of vitamin D. Clin Biochem Rev 2010;31:129. [PMC free article] [PubMed] [Google Scholar]
  • 7.Pfeifer M, Begerow B, Minne HW. Vitamin D and muscle function. Osteoporosis Int 2002;13:187–94. doi: 10.1007/s001980200012 [DOI] [PubMed] [Google Scholar]
  • 8.Cohen-Lahav M, Douvdevani A, Chaimovitz C, Shany S. The anti-inflammatory activity of 1,25-dihydroxyvitamin D3 in macrophages. J Steroid Biochem 2007;103:558–62 doi: 10.1016/j.jsbmb.2006.12.093 [DOI] [PubMed] [Google Scholar]
  • 9.Agbalalah T, Hughes SF, Freeborn EJ, Mushtaq S. Impact of vitamin D supplementation on endothelial and inflammatory markers in adults: A systematic review. J Steroid Biochem 2017. doi: 10.1016/j.jsbmb.2017.01.015 [DOI] [PubMed] [Google Scholar]
  • 10.Tarcin O, Yavuz DG, Ozben B, Telli A, Ogunc AV, Yuksel M, et al. Effect of vitamin D deficiency and replacement on endothelial function in asymptomatic subjects. J Clin Endocrinol Metab 2009;94:4023–30. doi: 10.1210/jc.2008-1212 [DOI] [PubMed] [Google Scholar]
  • 11.Ferder M, Inserra F, Manucha W, Ferder L. The world pandemic of vitamin D deficiency could possibly be explained by cellular inflammatory response activity induced by the renin- angiotensin system. Am J Physiol Cell Physiol 2013;304: C1027–39. doi: 10.1152/ajpcell.00403.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Vu D, Ong JM, Clemens TL, Kern PA. 1,25-Dihydroxyvitamin D induces lipoprotein lipase expression in 3T3-L1 cells in association with adipocyte differentiation. Endocrinology 1996;137:1540–4. doi: 10.1210/endo.137.5.8612483 [DOI] [PubMed] [Google Scholar]
  • 13.Bacha F, Arslanian SA. Race or vitamin D: A determinant of intima media thickness in obese adolescents? Pediatric diabetes 2017;18:619–21. doi: 10.1111/pedi.12472 [DOI] [PubMed] [Google Scholar]
  • 14.Pacifico L, Anania C, Osborn JF, et al. Low 25(OH)D3 levels are associated with total adiposity, metabolic syndrome, and hypertension in Caucasian children and adolescents. Eur J Endocrinol 2011;165:603–11. doi: 10.1530/EJE-11-0545 [DOI] [PubMed] [Google Scholar]
  • 15.Cheraghi N, Dai H, Raghuveer G. Vitamin D deficiency is associated with atherosclerosis-promoting risk factor clustering but not vascular damage in children. Med Sci Monit 2012;18:CR687–92. doi: 10.12659/msm.883593 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Singh J, Merrill ED, Sandesara PB, Schoeneberg L, Dai H, Raghuveer G. Vitamin D, low-lrade inflammation and cardiovascular risk in young children: a pilot study. Pediatr Cardiol 2015;36:1338–43 doi: 10.1007/s00246-015-1162-0 [DOI] [PubMed] [Google Scholar]
  • 17.Murni IK, Sulistyoningrum DC, Oktaria V. Association of vitamin D deficiency with cardiovascular disease risk in children: implications for the Asia Pacific Region. Asia Pac J Clin Nutr 2016;25(Suppl 1):S8–19. doi: 10.6133/apjcn.122016.s1 [DOI] [PubMed] [Google Scholar]
  • 18.de Onis M, Onyango AW, Borghi E, Siyam A, Nishida C, Siekmann J. Development of a WHO growth reference for school-aged children and adolescents. Bull World Health Organ 2007;85:660–7. doi: 10.2471/blt.07.043497 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Centers for Disease Control and Prevention. 2000 CDC Growth charts for the United States: Methods and Development. Vital and Health Statistics 2002; series 11(246). [PubMed] [Google Scholar]
  • 20.Cole TJ, Lobstein T. Extended international (IOFT) body mass index cut-offs for thinness, overweight and obesity. Pediatr Obes 2012;7:284–94. doi: 10.1111/j.2047-6310.2012.00064.x [DOI] [PubMed] [Google Scholar]
  • 21.IDF. The IDF consensus definition of the Metabolic Syndrome in children and adolescents. 2007
  • 22.Flynn JT, Kaelber DC, Baker-Smith CM, Blowey D, Carroll AE, Daniels SR, et al. Clinical practice guideline for screening and management of high blood pressure in children and adolescents. Pediatrics. 2017;140:e20171904. doi: 10.1542/peds.2017-1904 [DOI] [PubMed] [Google Scholar]
  • 23.Kimm H, Lee SW, Lee HS, Shim KW, Cho CY, Yun JE et al. Associations between lipid measures and Metabolic Syndrome, insulin resistance and adiponectin: usefulness of lipid ratios in Korean men and women. Circ J 2010;74: 931–7. doi: 10.1253/circj.cj-09-0571 [DOI] [PubMed] [Google Scholar]
  • 24.Keskin M, Kurtoglu S, Kendirci M, Atabek E, Yazici C. Homeostasis model assessment s more reliable than the fasting glucose/insulin ratio and quantitative insulin sensitivity check index for assessing insulin resistance among obese children and adolescents. Pediatrics 2005;115:e500–3. doi: 10.1542/peds.2004-1921 [DOI] [PubMed] [Google Scholar]
  • 25.McLaughlin T, Abbasi F, Cheal K, Chu J, Lamendola C, Reaven G. Use of metabolic markers to identify overweight individuals who are insulin resistant. Ann Intern Med 2003;139:802–9. doi: 10.7326/0003-4819-139-10-200311180-00007 [DOI] [PubMed] [Google Scholar]
  • 26.Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary Report. Pediatrics 2011; 128 (Suppl 5). [DOI] [PMC free article] [PubMed]
  • 27.Elhoseiny SM, Morgan DS, Rabie AM, Bishay ST. Vitamin D Receptor (VDR) gene polymorphisms (FokI, BsmI) and their relation to vitamin D status in pediatrics beta Thalassemia major. Indian J Hematol Blood Transfus 2016;32:228–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Stein JH, Korcarz CE, Hurst RT, Lonn E, Kendall CB, Mohler ER, et al. Use of carotid ultrasound to identify subclinical vascular disease and evaluate cardiovascular disease risk: a consensus statement from the American Society of Echocardiography Carotid Intima-Media Thickness Task Force. Endorsed by the Society for Vascular Medicine. J Am Soc Echocardiogr. 2008;21:93–111. doi: 10.1016/j.echo.2007.11.011 [DOI] [PubMed] [Google Scholar]
  • 29.Murni IK, Sulistyoningrum DC, Susilowati R, Julia M. Risk of metabolic syndrome and early vascular markers for atherosclerosis in obese Indonesian adolescents. Paediatr Int Child Health 2019. doi: 10.1080/20469047.2019.1697568 [DOI] [PubMed] [Google Scholar]
  • 30.Ganji V, Zhang X, Shaikh N, Tangpricha V. Serum 25-hydroxyvitamin D concentrations are associated with prevalence of metabolic syndrome and various cardiometabolic risk factors in US children and adolescents based on assay-adjusted serum 25-hydroxyvitamin D data from NHANES 2001–2006. Am J Clin Nutr 2011;94:225–33. doi: 10.3945/ajcn.111.013516 [DOI] [PubMed] [Google Scholar]
  • 31.Lee DY, Kwon AR, Ahn JM, et al. Relationship between serum 25-hydroxyvitamin D concentration and risks of metabolic syndrome in children and adolescents from Korean National Health and Nutrition Examination survey 2008–2010. Ann Pediatr Endocrinol Metab 2015;20:46–52. doi: 10.6065/apem.2015.20.1.46 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Soesanti F, Pulungan A, Tridjaja B, Batubara J. Vitamin D profile in healthy children aged 7–12 years old in Indonesia. Int J Pediatr Endocrinol 2013:P167. [Google Scholar]
  • 33.McGill HC Jr. The pathogenesis of atherosclerosis. Clin Chem 1988;34(8B):B33–9 [PubMed] [Google Scholar]
  • 34.Napoli C, Pignalosa O, de Nigris F, Sica V. Childhood infection and endothelial dysfunction: a potential link in atherosclerosis? Circulation 2005;111:1568–70. doi: 10.1161/01.CIR.0000161816.52136.66 [DOI] [PubMed] [Google Scholar]
  • 35.Strong JP, Mc GH Jr. The natural history of coronary atherosclerosis. Am J Pathol 1962;40:37–49. [PMC free article] [PubMed] [Google Scholar]
  • 36.Mao PJ, Zhang C, Tang L, et al. Effect of calcium or vitamin D supplementation on vascular outcomes: a meta-analysis of randomized controlled trials. Int J Cardiol 2013;169:106–11. doi: 10.1016/j.ijcard.2013.08.055 [DOI] [PubMed] [Google Scholar]
  • 37.Somjen D, Weisman Y, Kohen F, et al. 25-hydroxyvitamin D3-1alpha-hydroxylase is expressed in human vascular smooth muscle cells and is upregulated by parathyroid hormone and estrogenic compounds. Circulation 2005;111:1666–71. doi: 10.1161/01.CIR.0000160353.27927.70 [DOI] [PubMed] [Google Scholar]
  • 38.Susilowati R, Sulistyoningrum DC, Witari NP, Huriyati E, Luglio HF, Julia M. Sexual dimorphism in interleukin 17A and adipocytokines and their association with insulin resistance among obese adolescents in Yogyakarta, Indonesia. Asia Pac J Clin Nutr 2016;25(Suppl 1):S93–101. doi: 10.6133/apjcn.122016.s13 [DOI] [PubMed] [Google Scholar]
  • 39.Li YC, Kong J, Wei M, Chen ZF, Liu SQ, Cao LP. 1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system. J Clin Invest 2002;110:229–38. doi: 10.1172/JCI15219 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Berenson GS, Srinivasan SR, Bao W, Newman WP, Tracy RE, Wattigney WA. Association between multiple cardiovascular risk factors and atherosclerosis in children and young adults. The Bogalusa Heart Study. N Engl J Med 1998;338:1650–6. doi: 10.1056/NEJM199806043382302 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Jonathan M Peterson

30 Jul 2020

PONE-D-20-19921

SEX DIFFERENCES IN THE ASSOCIATION OF VITAMIN D AND METABOLIC RISK FACTORS WITH CAROTID INTIMA-MEDIA THICKNESS IN OBESE ADOLESCENTS

PLOS ONE

Dear Dr. Murni,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

While the study had a lot of merit, there were a number of issues that need to be addressed.  First their were significant grammatically errors throughout the text. Second, the data presented in the tables is unclear (what is being presented, skewed data is not indicated, and what was being compared). Third, why was Vitamin D not examined as a continuous variable? Also, from the raw data it appears that blood pressure was measured, why was this not included in the analysis? Lastly, as you did not find a association between vitD and CIMT, why was the analysis not extended to look at vitamin D to other metabolic risk factors measured.

Please submit your revised manuscript by Sep 13 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Jonathan M Peterson, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.  

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

●      The name of the colleague or the details of the professional service that edited your manuscript

●      A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

●      A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

3. In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants. Please ensure you have provided sufficient details to replicate the analyses such as: a) the recruitment date range (month and year), b) a description of any inclusion/exclusion criteria that were applied to participant recruitment, c) a table of relevant demographic details, d) a statement as to whether your sample can be considered representative of a larger population, e) a description of how participants were recruited, and f) descriptions of where participants were recruited and where the research took place.

4. Thank you for stating in your Funding Statement:

 "The study was partly funded by the Indonesian Pediatric Society and Frisian Flag Indonesia (355/Legal/FFI/XII/2014)".

i) Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

ii) Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Additional Editor Comments (if provided):

While the study had a lot of merit, there were a number of issues that need to be addressed. First their were significant grammatically errors throughout the text. Second, the data presented in the tables is unclear (what is being presented, skewed data is not indicated, and what was being compared). Third, why was Vitamin D not examined as a continuous variable? Also, from the raw data it appears that blood pressure was measured, why was this not included in the analysis? Lastly, as you did not find a association between vitD and CIMT, why was the analysis not extended to look at vitamin D to other metabolic risk factors measured.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Paper has lot of grammatical errors and flow is very limited

Need to assess high sensitivity CRP/?cardiac risk/LVMI and 24 hr ABPM or BP and would have correlated these parameters with those variables

Vitamin d level should be correlated with these parameters as we do not know what level is optimal one

urine albumin to creatinine ratio should reassesses

I will assess height and growth parameters 'also

need to develop or assess cardiovascular risk score like in adult to assess risk prediction

we should look into family history

It is association not causation

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Oct 15;16(10):e0258617. doi: 10.1371/journal.pone.0258617.r002

Author response to Decision Letter 0

15 Sep 2020

Thank you for the constructive questions and feedbacks. Herewith our response to Reviewers.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response to the Reviewer:

Thank you, noted.

2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

● The name of the colleague or the details of the professional service that edited your manuscript

● A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

● A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

Response to the Reviewer:

The manuscript had been checked for the grammars and spellings by:

Erik Christopher Hookom, BA, Med, TEFL

Office of Research and Publication (ORP)

Faculty of Medicine, Public Health, and Nursing Universitas Gadjah Mada

Administration Building 2nd Floor

Phone: 0274 560300 ext 205

Email: orp.fm@ugm.ac.id; echookom@gmail.com

A copy of manuscript showing track changes and a clean copy of the edited manuscript have been uploaded as a supporting information file and the new manuscript file, respectively.

3. In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants. Please ensure you have provided sufficient details to replicate the analyses such as: a) the recruitment date range (month and year), b) a description of any inclusion/exclusion criteria that were applied to participant recruitment, c) a table of relevant demographic details, d) a statement as to whether your sample can be considered representative of a larger population, e) a description of how participants were recruited, and f) descriptions of where participants were recruited and where the research took place.

Response to the Reviewer:

Thank you for the feedbacks. The recruitment process was done by screening for adolescents with obesity in seven public and three private high schools in the city of Yogyakarta, a city in the Southern part of Java, Indonesia. The participants were recruited at their schools and we screened 4268 students from 10 schools in Yogyakarta using three body mass index reference cut-off points: World Health Organization (WHO), Centre for Disease Control and Prevention (CDC), and International Obesity Task Force (IOTF). The screening was done from January to February 2016. We identified 298 (7%) adolescents classified as obese based on all those three obesity criteria. Of those, 229 (76.8%) obese adolescents agreed to participate in the next step of the study, i.e. assessment of their metabolic risk. The blood collection was taken at their schools, while the assessment of carotid intima media thickness was performed at the Dr Sardjito Hospital, Yogyakarta. These data collection was done from March to October 2016.

Inclusion criteria were obese children aged 15 to less than 18 years. We excluded children with diabetes mellitus, renal disease, cardiovascular disease, any history of any systemic disease or history of current steroid use. This study included 156 (out of 229) obese adolescents who had data on CIMT. We considered that the sample of this study could be considered representative of a larger population.

There were no differences in metabolic risk parameters between adolescents who had their CIMT measured versus those who had not (Appendix).

4. Thank you for stating in your Funding Statement:

"The study was partly funded by the Indonesian Pediatric Society and Frisian Flag Indonesia (355/Legal/FFI/XII/2014)".

i) Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Response to the Reviewer:

Thank you for the feedback. Regarding the funding of this study, we declare that the study was funded by the Indonesian Pediatric Society and Frisian Flag Indonesia (355/Legal/FFI/XII/2014). There was no additional external funding received for this study.

ii) Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

Response to the Reviewer:

Thank you, the amended Funding Statement has been included in the cover letter.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Response to the Reviewer:

Done

Additional Editor Comments (if provided):

While the study had a lot of merit, there were a number of issues that need to be addressed. First their were significant grammatically errors throughout the text. Second, the data presented in the tables is unclear (what is being presented, skewed data is not indicated, and what was being compared). Third, why was Vitamin D not examined as a continuous variable? Also, from the raw data it appears that blood pressure was measured, why was this not included in the analysis? Lastly, as you did not find a association between vitD and CIMT, why was the analysis not extended to look at vitamin D to other metabolic risk factors measured.

While the study had a lot of merit, there were a number of issues that need to be addressed. First their were significant grammatically errors throughout the text.

Response to the Editor:

The manuscript had been checked grammatically by a professional scientific editing service and this has been stated in the cover letter.

Second, the data presented in the tables is unclear (what is being presented, skewed data is not indicated, and what was being compared).

Response to the Reviewer:

Thank you for the feedback. We have added explanation on the dependent and independent variables on the Tables and the skewed data have been clearly indicated.

Table 1 describes the characteristics of obese adolescents from Yogyakarta stratified by sex. Normally distributed and skewed data are presented as mean (SD) and median (Q1;Q3), respectively. Normally distributed data are compared using independent sample t-test, while skewed data are compared using independent-samples Mann-Whitney U test. Categorical data are presented as n (%) and compared using chi-square tests unless otherwise indicated.

Table 2 describes sex differences in the association of vitamin D and metabolic risk factors with carotid intima-media thickness in obese adolescents from Yogyakarta, Indonesia.

The dependent variable was CIMT as a marker of early vascular impairment. The independent variables were vitamin D and other metabolic risk factors including waist-to-hip ratio (marker of abdominal obesity), blood glucose, HbA1c (impaired glucose metabolism), fasting insulin, HOMA-IR (insulin resistance), LDL and HDL cholesterol, triglyceride (impaired lipid metabolism), blood pressure, smoking and family history of hypertension.

Table 2 shows that overall, elevated HOMA-IR, total cholesterol, LDL-cholesterol and triglyceride levels were associated with greater odds of elevated CIMT. The associations persisted after adjustment for age, sex and second-hand smoke exposure. However, after the data were analysed within each sex, similar results were only observed in boys. None of the risk factors were associated with CIMT in girls.

Third, why was Vitamin D not examined as a continuous variable?

Response to the Reviewer:

Thank you for the feedback. We actually are interested in looking at the clinical consequences of deficiency or insufficiency cut-offs of vitamin D status with the carotid intima media thickness as a measure of vascular thickness (a subclinical atherosclerosis in children). We used different cut off: Vitamin D level ≤30 ng/mL or ≤20 ng/mL. A categorisation of continuous data of vitamin D level was used in this study because it is easier to interpret and more feasible to present in clinical practice. It is helpful to label children and adolescents as having deficient, insufficient or normal vitamin D level. We hope that this paper is easier for clinicians to read and understand in order to make clinical decision making where decisions typical are categorical in nature.

Also, from the raw data it appears that blood pressure was measured, why was this not included in the analysis?

Response to the Editor:

We have included the analysis of blood pressure (as one of independent variables) and CIMT (as a dependent variable or outcome) at Table 2.

Lastly, as you did not find a association between vitD and CIMT, why was the analysis not extended to look at vitamin D to other metabolic risk factors measured.

Response to the Editor:

Thank you for the feedback. It seems like the introduction section of the paper was not clear enough to highlight why we are interested in looking at CIMT as predicted by vitamin D status independent of other predictors of CIMT.

A paragraph has been added in the Introduction to further clarify the aim of the study.

Carotid intima-media thickness (CIMT) is regarded as a reliable marker of subclinical atherosclerosis since increased CIMT can reflect an increase in arterial wall thickness including in children and adolescent (Atabek et al, 2014). Increased CIMT has been predicted in children with obesity, familial hypercholesterolemia, type 1 diabetes, hypertension (Giladini et al, 2011) and vitamin D deficiency (Atabek et al, 2014). In this study, we are particularly interested in looking at CIMT as predicted by vitamin D status independent of other predictors of CIMT.

The dependent variable was CIMT as a marker of early vascular impairment. The independent variables were vitamin D and other metabolic risk factors including waist-to-hip ratio (marker of abdominal obesity), blood glucose, HbA1c (impaired glucose metabolism), fasting insulin, HOMA-IR (insulin resistance), LDL and HDL cholesterol, triglyceride (impaired lipid metabolism), blood pressure, smoking and family history of hypertension.

The association of vitamin D and other metabolic risk factors with CIMT was explored using logistic regression and presented as odds ratio (95% confidence interval). Each factor was tested in a separate regression model. Models that were significantly associated with elevated CIMT, were re-tested with adjustment for age, sex and secondary smoke exposures. All analyses were performed on a sample as a whole and also stratified by sex based on biological differences in body fat accumulation and potentially vitamin D levels between boys and girls.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Paper has lot of grammatical errors and flow is very limited

Need to assess high sensitivity CRP/?cardiac risk/LVMI and 24 hr ABPM or BP and would have correlated these parameters with those variables

Vitamin d level should be correlated with these parameters as we do not know what level is optimal one

urine albumin to creatinine ratio should reassesses

I will assess height and growth parameters 'also

need to develop or assess cardiovascular risk score like in adult to assess risk prediction

we should look into family history

It is association not causation

Need to assess high sensitivity CRP/?cardiac risk/LVMI and 24 hr ABPM or BP and would have correlated these parameters with those variables. Vitamin d level should be correlated with these parameters as we do not know what level is optimal one

Response to the Reviewer:

Thank you for the feedback. In this study, we particularly interested in looking at CIMT as predicted by vitamin D status independent of other predictors of CIMT. We are interested in looking at the clinical consequences of deficiency or insufficiency cut-offs of vitamin D status with the carotid intima media thickness as a measure of vascular thickness (a subclinical atherosclerosis in children).

We did not have data on hsCRP. But we did analyses on the association between CIMT (as an outcome) and vitamin D and other cardiovascular and metabolic risk factors including abdominal obesity (waist to hip ratio), blood glucose, HbA1c (glucose metabolism impairment), fasting insulin, HOMA-IR (insulin resistance), LDL and HDL cholesterol, triglyceride (dyslipidaemia), blood pressure, smoking, and family history of hypertension. We have added a paragraph in the Method section to clarify this.

We have added analysis on blood pressure and CIMT in the Table 2. But we did not include the analysis of LVMI in this study because we will do in another paper.

urine albumin to creatinine ratio should reassesses

Response to the Reviewer:

Thank you for the feedback. We did not have data on albumin and creatinine ratio.

I will assess height and growth parameters 'also

Response to the Reviewer:

Thank you for the feedback. We have analysis the height in term of waist-to-hip ratio between boys and girls in the Table 1 and the association between high waist-to-hip ratio with CIMT in the Table 2. But we did not analyse the association between growth with CIMT. We did not have the data of growth because of the nature of cross sectional study design.

need to develop or assess cardiovascular risk score like in adult to assess risk prediction

we should look into family history

Response to the Reviewer:

Thank you for the feedback. In this study we did not perform a cardiovascular risk score, we only assessed whether vitamin D and other metabolic risk factors predicted the increased CIMT without creating a score. But we have included a family history of hypertension as one of predictors of increased CIMT in this study.

It is association not causation

Response to the Reviewer:

Thank you, noted.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (28.4KB, docx)

Decision Letter 1

Jonathan M Peterson

23 Nov 2020

PONE-D-20-19921R1

SEX DIFFERENCES IN THE ASSOCIATION OF VITAMIN D AND METABOLIC RISK FACTORS WITH CAROTID INTIMA-MEDIA THICKNESS IN OBESE ADOLESCENTS

PLOS ONE

Dear Dr. Murni,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

First I would like to apologize for the long delay, it was unfortunately as we had significant issues securing a reviewer for the manuscript.

I agree with the new reviewers comments that the manuscript is much improved, but analysis of VitD as a continuous variable should also be included. Regarding the table legends, it is still unclear what is being compared.  For table 1 the p value appears to be reporting significance comparing boys and girls? However, the p-values reported for table 2 are difficult to discern and are not explained clearly in the legend.

Please submit your revised manuscript by Jan 07 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Jonathan M Peterson, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

First I would like to apologize for the long delay, it was unfortunately as we had significant issues securing a reviewer for the manuscript.

I agree with the new reviewers comments that the manuscript is much improved, but analysis of VitD as a continuous variable should also be included. Regarding the table legends, it is still unclear what is being compared. For table 1 the p value appears to be reporting significance comparing boys and girls? However, the p-values reported for table 2 are difficult to discern and are not explained clearly in the legend.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The carotid intima-media thickness (CIMT) is already well studied in children and adolescents, including assessing the relationship with low levels of vitamin D 25-OH.

The present study provides data from Indonesian adolescents, which is important to verify possible differences in this specific population.

The grammatical revision of the text really improved its quality. However, some aspects remain deficient:

1. Although the analysis of vitamin D as a categorical variable had been justified, it would be important, which is not so difficult, also analyze it as a continuous variable. Even if the result is negative, the analysis will be more complete. After all, the title, and the entire text focus on vitamin D. Otherwise, I suggest expanding the discussion to the other parameters evaluated, drawing attention to the positive findings. (Osika W, Dangardt F, Montgomery SM, Volkmann R, Gan LM, Friberg P. Sexual differences in the intimate peripheral artery, mean and thickness of the intimate media in children and adolescents. Atherosclerosis. 2009 March; 203 (1): 172-7 .)

2. Verification of normality uses the Kolmogorov-Smirnov test. In the text, “Smirnov” was replaced by a brand of vodka.

3. It is necessary to exclude the form of data presentation in the first columns, leaving only the name of the variable and the unit. The current format is redundant (the information is already described below the tables) and makes it difficult to understand.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Oct 15;16(10):e0258617. doi: 10.1371/journal.pone.0258617.r004

Author response to Decision Letter 1

6 Jan 2021

Response to Reviewer

Thank you for the constructive questions and feedbacks.

Additional Editor Comments (if provided):

First I would like to apologize for the long delay, it was unfortunately as we had significant issues securing a reviewer for the manuscript.

I agree with the new reviewers comments that the manuscript is much improved, but analysis of VitD as a continuous variable should also be included. Regarding the table legends, it is still unclear what is being compared. For table 1 the p value appears to be reporting significance comparing boys and girls? However, the p-values reported for table 2 are difficult to discern and are not explained clearly in the legend.

Thank you. The analysis of vitamin D levels as a continuous variable has been provided below:

Table 2. Correlations between of vitamin D levels and other metabolic risk factors to predict carotid intima-media thickness in obese adolescents (attached)

We found no correlation between vitamin D levels and CIMT, but we found a positive correlation between LDL and total cholesterols with CIMT.

We also provide Table 3 showing the association of vitamin D levels and other metabolic risk factors to predict carotid intima-media thickness between boy and girl obese adolescents. Normally distributed continuous data are compared using independent sample t-test, while skewed data are compared using independent-samples Mann-Whitney U test. Categorical data are presented as n (%) and compared using chi-square tests unless otherwise indicated.

p value in the previous table 1 reported the significance comparing boys and girls.

Odds ratio with its 95% CI and p value in the previous table 2 (now as Table 4) reported the significance of association of vitamin D levels and other metabolic risk factors to predict carotid intima-media thickness comparing boys and girls using logistic regression analysis.

Reviewer #2: The carotid intima-media thickness (CIMT) is already well studied in children and adolescents, including assessing the relationship with low levels of vitamin D 25-OH.

The present study provides data from Indonesian adolescents, which is important to verify possible differences in this specific population.

The grammatical revision of the text really improved its quality. However, some aspects remain deficient:

1. Although the analysis of vitamin D as a categorical variable had been justified, it would be important, which is not so difficult, also analyze it as a continuous variable. Even if the result is negative, the analysis will be more complete. After all, the title, and the entire text focus on vitamin D. Otherwise, I suggest expanding the discussion to the other parameters evaluated, drawing attention to the positive findings. (Osika W, Dangardt F, Montgomery SM, Volkmann R, Gan LM, Friberg P. Sexual differences in the intimate peripheral artery, mean and thickness of the intimate media in children and adolescents. Atherosclerosis. 2009 March; 203 (1): 172-7 .)

Response to the Reviewer:

Thank you. The analysis of vitamin D levels as a continuous variable has been provided in the new version of Table 2 below:

Table 2. Correlations between of vitamin D levels and other metabolic risk factors to predict carotid intima-media thickness in obese adolescents (attached)

We found no correlation between vitamin D and CIMT, but we found a positive correlation between LDL and total cholesterols with CIMT.

We also provide Table 3 showing the association of vitamin D levels and other metabolic risk factors to predict carotid intima-media thickness between boy and girl obese adolescents. Normally distributed continuous data are compared using independent sample t-test, while skewed data are compared using independent-samples Mann-Whitney U test. Categorical data are presented as n (%) and compared using chi-square tests unless otherwise indicated.

The new table 4 showed association of vitamin D levels and other metabolic risk factors to predict carotid intima-media thickness comparing obese adolescent boys and girls using logistic regression analysis.

2. Verification of normality uses the Kolmogorov-Smirnov test. In the text, “Smirnov” was replaced by a brand of vodka.

Thank you, we have revised accordingly.

3. It is necessary to exclude the form of data presentation in the first columns, leaving only the name of the variable and the unit. The current format is redundant (the information is already described below the tables) and makes it difficult to understand.

Thank you, we have revised accordingly as suggested.

Attachment

Submitted filename: Response to Reviewer.docx

Click here for additional data file. (23KB, docx)

Decision Letter 2

Raffaella Buzzetti

15 Sep 2021

PONE-D-20-19921R2SEX DIFFERENCES IN THE ASSOCIATION OF VITAMIN D AND METABOLIC RISK FACTORS WITH CAROTID INTIMA-MEDIA THICKNESS IN OBESE ADOLESCENTSPLOS ONE

Dear Dr. Murni,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 30 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Raffaella Buzzetti, M.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: Minor revisions are needed:

table 3: change the title of table 3. Authors did not check for "associations" but differences in patients with elevated CIMT and not elevated CIMT. Further, do not repeat statistical methods in the legend of the table, please revise also the sentence in tesxt (LDL cholesterol was associated with carotid intima media thickness among obese adolescent boys). Boys with elevated CIMT showed increased values of LDL cholesterol it is

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Oct 15;16(10):e0258617. doi: 10.1371/journal.pone.0258617.r006

Author response to Decision Letter 2

29 Sep 2021

Reviewer #3: Minor revisions are needed:

table 3: change the title of table 3. Authors did not check for "associations" but differences in patients with elevated CIMT and not elevated CIMT. Further, do not repeat statistical methods in the legend of the table, please revise also the sentence in tesxt (LDL cholesterol was associated with carotid intima media thickness among obese adolescent boys). Boys with elevated CIMT showed increased values of LDL cholesterol it is

Response to Reviewer:

Thank you so much for the advice, we have revised accordingly as suggested in the manuscript.

Attachment

Submitted filename: Response to Reviewers comment.docx

Click here for additional data file. (15.2KB, docx)

Decision Letter 3

Raffaella Buzzetti

4 Oct 2021

SEX DIFFERENCES IN THE ASSOCIATION OF VITAMIN D AND METABOLIC RISK FACTORS WITH CAROTID INTIMA-MEDIA THICKNESS IN OBESE ADOLESCENTS

PONE-D-20-19921R3

Dear Dr. Murni,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Raffaella Buzzetti, M.D.

Academic Editor

PLOS ONE

Acceptance letter

Raffaella Buzzetti

7 Oct 2021

PONE-D-20-19921R3

Sex differences in the association of vitamin D and metabolic risk factors with carotid intima-media thickness in obese adolescents

Dear Dr. Murni:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Raffaella Buzzetti

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Appendix. Characteristics of obese adolescents who had and had not CIMT measured.

    (DOCX)

    Click here for additional data file. (14.7KB, docx)
    S1 Fig. Prevalence of increased carotid intima media thickness across vitamin D status among obese adolescents.

    (TIFF)

    Click here for additional data file. (217.8KB, tiff)
    S1 Data. Dataset of vitamin D and CIMT.

    (XLSX)

    Click here for additional data file. (134.3KB, xlsx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (28.4KB, docx)
    Attachment

    Submitted filename: Response to Reviewer.docx

    Click here for additional data file. (23KB, docx)
    Attachment

    Submitted filename: Response to Reviewers comment.docx

    Click here for additional data file. (15.2KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information file.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES