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ORIGINAL ARTICLE
Received 23 December 2014; received in revised form 26 February 2015; accepted 1 April 2015
KEYWORDS
antioxidant;
atherosclerosis;
inflammation;
scavenging receptor
* Corresponding author. Division of Nephrology, Department of Child Health, Faculty of Medicine, Diponegoro University/Dr. Kariadi
Hospital, Jalan Dokter Sutomo Number 16, Semarang, Central Java 50244, Indonesia.
E-mail address: omegamellyana@gmail.com (O. Mellyana).
http://dx.doi.org/10.1016/j.bgm.2015.04.001
2214-0247/Copyright 2015, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
126
O. Mellyana et al.
the levels of ox-LDL, sVCAM-1, and NOx were decreased after treatment with a combined supplementation of vitamins C and E, but there was no statistical difference (p > 0.05). After
treatment, there was an increase in the level of sCD36 in both groups, although this was not
significantly different (p > 0.05). The level of ox-LDL was significantly lower in the
remission-idiopathic NS (remission-INS) group compared with the nonremission-INS group
(p < 0.05). The levels of ox-LDL and sVCAM-1 were significantly lower in the remissiontreated group than in the nonremission-treated group (p < 0.05). In conclusion, the combined
supplementation of vitamins C and E cannot modify the ox-LDL, sCD36, sVCAM-1, and NOx
levels in children with INS. In remission cases, the combined supplementation of vitamins C
and E reduces the ox-LDL and sVCAM-1 levels.
Copyright 2015, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier
Taiwan LLC. All rights reserved.
Introduction
Idiopathic factor is a common cause of nephrotic syndrome
(NS) in children.1 This pathomechanism can be classified
directly from kidney disease (primary NS/idiopathic) or
indirectly via conditions in other parts of the body affecting
the kidney (secondary NS).2,3 Persistent NS is frequently
associated with abnormal lipid profiles, including elevated
levels of total lipids [cholesterol and triglycerides (TGs)],
and an often enormous increase in cholesterol in the lowdensity lipoprotein (LDL)- and very-low-density lipoprotein-cholesterol fraction. Conversely, the concentration of
high-density lipoprotein (HDL)-cholesterol (HDL-C) is
normal or even decreased. This condition constitutes a risk
factor for early atherosclerosis.4,5
Oxidative modification of LDL (ox-LDL) is a central
pathogenesis of atherosclerosis.6 ox-LDL is an early event of
subsequent pathogenetic conditions and is associated with
carotid intimaemedia thickening; release of unstable plaques in vascular, brachial, and coronary endothelial
dysfunction; and coronary artery disease.7 When ox-LDL
loses its ability to bind with LDL receptors, it will interact
with proteins called scavenger receptors.8 CD36, originally
described as a platelet receptor glycoprotein (88 kDa),
belongs to the class B scavenger receptor family.9,10 A soluble form of CD36 (sCD36) can be identified in human
plasma, and its level is shown to increase in atherogenesis.11 A recent study showed that sCD36 is not a proteolytic
product, but is rather associated with a specific subset of
circulating microparticles (MPs) that can readily be
analyzed. These MPs primarily originated from platelets.12
Previous studies have shown that oxidative modification
of LDL was involved in NS. The oxidized form of lipoprotein
levels was significantly higher in acute-period NS and
remission NS compared with control.13 Plasma malondialdehyde as a lipid peroxidation marker was found to increase
in patients with NS compared with controls. Plasma ascorbic
acid was decreased in the nephrotic group than in healthy
controls.14,15 The ratio of vitamin E to LDL was reduced in the
active stage compared with healthy controls.16
Nitric oxide synthase (NOS) is required for the synthesis
of nitric oxide (NO). All three NOS isoforms can be
expressed in the kidney.17 The association of NO level with
idiopathic NS (INS) was inconsistent. One study showed an
increased level of urinary nitrite in children with NS.18
Another study found that the eNOS4 gene polymorphisms
Definitions
Primary (idiopathic) NS was defined by heavy proteinuria
(urinary protein > 50 mg/kg/d or dipstick 2), hypoalbuminemia (serum albumin < 2.5 g/dL), and hypercholesterolemia (serum cholesterol > 5.72 mmol/L or >
200 mg/dL), with or without edema. The initial treatment
includes oral administration of prednisone (2 mg/kg/d;
Blood samples
Blood sample was drawn from an antecubital vein into 10mL serum Vacutainer tubes. After approximately 45 minutes, the tubes were centrifuged at 3000 rpm (5000g) for
10 minutes at room temperature. Serum was separated
from blood cells and stored at 20 C until further analysis.
127
Ethics
Human experimental procedures were approved by the
Institutional Ethics Committee of University of Diponegoro/
Dr. Kariadi Hospital, Semarang, Central Java, Indonesia.
Statistical analysis
Data are presented as mean standard deviation and the
differences between groups were analyzed using independent sample t test for normal distribution or ManneWhitney
U test for abnormal distribution with IBM SPSS for Windows
version 20.0 statistical package. A p value less than 0.05
was considered statistically significant.
Results
Patients
Six of the 42 children (14.3%) dropped out of the study.
Thus, we tested our hypothesis on 36 children (18 in each
group). The age, sex, frequency of normotension and prehypertension, and food record at baseline were not significantly different between groups (p > 0.05; Table 1). During
the observation period, there were no side effects due to
the vitamin or placebo treatment. In addition, the remission status, frequency of hypertension, and lipid profile
were not significantly different between the two groups
(p > 0.05; Table 2).
128
Table 1
O. Mellyana et al.
Baseline clinical characteristics and food record among patients.
Parameter
INS
Age (mo)
Sex (male/female)
Normotension
Prehypertension stage 2
Energy (kcal/d)
Carbohydrate (g)
Carbohydrate (%)
Lipid (g)
Lipid (%)
Protein (g)
Protein (%)
Cholesterol (mg)
PUFA (g)
MUFA (g)
SFA (g)
Fiber (g)
Vitamin E
Vitamin C
80.48 42.24
12/9
8 (38.1%)
13 (61.9%)
1452.6 2777.73
199.9 56.86
54.3 9.50
50.6 15.37
30.5 6.98
53.7 17.6
15.1 5.8
268.7 200.79
5.3 3.3
8.65 4.4
23.5 8.68
6.7 3.68
4.3 2.76
40.8 37.6
87.81 45.10
13/8
9 (42.9%)
12 (57.1%)
1358.8 308.67
186.7 37.75
55.9 8.38
45.94 17.00
28.9 6.79
52.5 19.6
15.3 3.5
196.8 149.34
7.0 4.14
9.89 5.39
21.7 7.97
84
3.7 1.65
62.5 45.26
0.458
0.753
0.753
0.753
0.307
0.378
0.575
0.359
0.456
0.840
0.308
0.704
0.195
0.633
0.491
0.263
0.439
0.061
INS Z idiopathic nephrotic syndrome; MUFA Z monounsaturated fatty acid; PUFA Z polyunsaturated fatty acid; SFA Z saturated fatty
acid.
Discussion
Hyperlipidemia results from hypoalbuminemia due to inhibition of the reaction (conversion of cholesterol of HDLs to
cholesterol esters) catalyzed by lecithin cholesterol acyltransferase and to an inhibition of HDL particle formation
Table 2
Parameter
INS
Remission:nonremission
Normotension
Prehypertension stage 2
Total cholesterol (mg/dL)
LDL (mg/dL)
HDL (mg/dL)
VLDL (mg/dL)
Triglyceride (mg/dL)
9:9
9 (50%)
9 (50%)
265.33 158.12
173.05 132.76
45.88 20.18
42.61 22.35
161.89 135.89
10:8
15 (83.4%)
3 (16.6%)
329.05 212.796
211.28 162.5
51.44 16.58
75.67 83.27
203.89 184.24
0.738
0.077
0.443
0.628
0.373
0.563
0.719
INS Z idiopathic nephrotic syndrome; LDL Z low-density lipoprotein; HDL Z high-density lipoprotein; VLDL Z very-low-density
lipoprotein.
129
Levels of ox-LDL, sVCAM-1, sCD36, and NOx before and after treatment.
Biomarkers
Before
ox-LDL (U/L)
sCD36 (ng/mL)
sVCAM-1 (ng/mL)
NOx (mM)
INS
125.2
56.9
1231.2
8.2
After
57.15
66.20
660.30
4.73
Before
76.7
61.7
1007.96
7.9
61.72
59.04
395.97
3.23
127.2
72.5
929.4
8.9
After
51.69
102.36
225.23
6.80
89.5
123.5
868.4
8.5
63.85
180.97
232.25
5.90
INS Z idiopathic nephrotic syndrome; NOx Z nitrite/nitrate oxide; ox-LDL Z oxidized low-density lipoprotein; sCD36 Z soluble cluster
of differentiation 36; sVCAM Z soluble vascular cells adhesion molecule.
Table 4
Levels of ox-LDL, sVCAM-1, sCD36, and NOx in remission and nonremission cases.
Biomarkers
Nonremission (n Z 9)
ox-LDL (U/L)
sVCAM-1 (ng/mL)
sCD36 (ng/mL)
NOx (mM)
INS
107.8
1072.0
55.8
8.2
66.1
454.7
51.70
3.4
Remission (n Z 9)
45.5
943.9
68.2
7.0
39.2*
342.3
69.4
3.1
Nonremission (n Z 8)
128.4
1060.3
85.8
8.2
59.3
199.2
150.0
5.6
Remission (n Z 10)
58.3
714.8
153.7
8.7
50.36**
109.8**
205.2
6.4
group (p < 0.05). This finding shows that the level of ox-LDL
will decrease with or without combined vitamin treatment.
Macrophage CD36 is surface molecule that is able to bind
and internalize LDL for the formation of foam cells.28,29
Elevated plasma sCD36 levels have been proposed to be a
marker of insulin resistance and atherosclerosis risk. In this
study, we found that the sCD36 level increased in both
groups, but this was not significantly different (p > 0.05).
We hypothesize that this increase might have been due to
the increased number of platelets and monocytes. A previous report showed that the circulating form of the sCD36
receptor is associated with MPs coming from platelets,
leukocytes, and endothelial cells upon receiving stimuli or
apoptosis signal.8 Besides, low-grade inflammation also induces the production of MPs.30e32
Asymmetric dimethylarginine (ADMA) is an endogenous
inhibitor of NO synthase. The inhibition of NO synthesis is
known to be associated with an increase in the expression
of endothelial adhesion molecule (sVCAM-1).33e36 In this
study, there was no significant difference in the levels of
NOx and sVCAM-1 between the two study groups (p > 0.05).
In a previous study of 15 teenage children with familial
hypercholesterolemia and familial combined hyperlipidemia, treatment with vitamins C (500 mg/d) and E (400 IU/
d) for 6 weeks did not have an effect on ADMA.26 In other
words, our study shows that the combined supplementation
of vitamins C and E cannot modulate the endothelial
dysfunction in patients with NS. Interestingly, the level of
sVCAM-1 was significantly decreased in the remissiontreated group, compared with the nonremission-treated
group (p < 0.05). This finding shows that the combined
supplementation of vitamins C and E modulates the endothelial dysfunction in patients with remission of NS.
In conclusion, the combined supplementation of vitamins C and E cannot modify the ox-LDL, sCD36, sVCAM-1,
Conflicts of interest
The authors declare that there are no conflicts of interest
regarding the publication of this article.
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