Research
THE EFFECT OF INTRAVENOUS VITAMIN C ON NITRIC OXIDE
SERUM LEVEL IN SEVERE BURN INJURY
(Experimental Study)
Bobby Swadharma Putra*; Lobredia Zarasade, Iswinarno Doso Saputro
Departement of Plastic & Reconstructive Surgery, Dr. Soetomo General Hospital, Airlangga University, School of Medicine,
Surabaya
ABSTRACT
Introduction: Nitric oxide (NO) has a potential rule in pathogenesis of causing systemic hypotension and apoptosis,
contributing to tissue damage and multiple organ failure in severe burn. Severe Burn increases the activity of the
Nitric Oxide Syntase (NOS) enzyme and Nitric Oxide release. Proinflamatory cytokines is inflammatory mediators
implicated in the induction and activation of NOS and NO release also. Vitamin C work as antioxidant by providing
hydrogen ions to free radical into stable molecule and blockade the activation of NF-kB so that suppresses
proinflamatory cytokines production and Nitric Oxide production can be inhibited. The purpose research To
review the effect of intravenous vitamin C3000mg on Nitric Oxide serum Level in severe burn injury patient post
fluid resuscitation.
.
Method: Randomized pre test and post test controlled group design experimental studied on 12 severe burns patient
at the Burn Unit of Dr. Soetomo Hospital in Surabaya who have been treated with baxter formula. Nitric Oxide
examined in 12 patient. Divided into 2 groups K 1 as the control group with vitaminC2x400mg/24 hour for 72 hours
; K2, administrered with intravenous vitamin C 3000mg for 72 hours. Nitric oxide then re-examined. Nitric Oxide
was taken from blood serum and observed by Grease method. The results were analyzed by Paired T-Test and
statistical assays, with reliability p < 0,05.
Result :Serum levels NO in the control group (K1) no significant increase in the serum levels of NO compared to
the first day of the fourth day (p=0,21) and no significant decrease in serum levels of NO in the first day than the
serum levels of NO on the fourth day (K2) (p=0,06). There is a statistically significant decrease in the levels of NO
in the group given intravenous vitamin 3000 mg/24 hours for 72 hours (K2) comparison with a control group (K1)
(p=0,02). From groups K 1as the control group there were no significant result in Blood Gas Analisis(BGA), Blood
Urea Nitrogen (BUN), and Serum Creatinine (SC). In the groups K 2 as administrered with intravenous vitamin C
3000 mg for 72 hours there were significant result in White Blood Cell (WBC) (p=0,01).
Conclusion: Research is a significant decrease in the levels of NO in the group that was given intravenous
vitamin C 3000mg /24hours for 72 hours comparison with a control group given intravenous vitamin 2x400mg /
24 hours for 72hours
Keywords: vitamin c, severe burn, nitric oxide
1
In severe burns nitric oxide (NO) has an
INTRODUCTION
important contribution, in which nitric oxide
Burn management and burn care is still a
(NO) is produced in large amounts by
challenge to the medical world, dur to the high
iNOSwhich
rate of its morbidity and mortality rates. In the
endothelial cells, and hepatic veins. Production
United
reported
of nitric oxide (NO) is increasing in severe burns
approximately 500,000 patients each year with
cause changes in cardiovascular function as a
mortality of about 3-4 thousand deaths / year in
result
2011. At Burn Unit at Dr. SoetomoHospital the
hipocontractility of blood vessels and heart
number of burn cases for one year (January 2011
muscle depression, also a circulatory failure may
to December 2011) are 167 cases, 94 cases
lead to a failure of an organ or a system called
(56.29%) were severe burns. Numbers of death
the
were11 people (19.54%) of all major burns.
(MODS) (Cakir, 2004).
States
burn
case
were
of
is
found
systemic
Multi-Organ
in
macrophages,
hypotension
Dysfunction
due
to
Syndrome
One of the systemic response caused by
The aim of this research is to determine
severe burns is the activation of NF-кB,
the effect of vitamin C 3000 mg / 24 hours for
transcription factor protein on macrophages
72 hours postbaxter resuscitation to the level of
which
NO in human blood who suffered severe burns.
will
increase
the
production
of
inflammatory mediators or proinflammatory
As an antioxidant, vitamin C can react
cytokines such as tumor necrosis factor (TNF-
with Reactive Oxygen Species (ROS) produced
α), interleukin (IL)) and interferon gamma (IFN-
by
γ). The increase of inflammatory mediators will
inflammatory phase, converting free radicals to a
enhance the expression of nitric oxide (NO) in
more inertform. Arachidonic acid cascade,
large numbers via activation of iNOS. iNOS is
which is activated by ROS will be cut so that the
expressed by several types of immune cells,
inflammatory reaction will stop. Therefore, the
especially macrophages.
anti-inflammatory effects allegedly associated
Nitric oxide (NO) is a cellular mediator,
produced by one of three NO synthase: nNOS(
neutrophils
and
macrophages
in
the
with vitamin C as an antioxidant nature (Lima,
2009).
neural nitric oxide syntase), eNO( endothelial
Vitamin C as an anti-inflammatory is
nitric oxide syntase) daniNOS ( inducible nitric
also capable of suppressing the activation of the
oxide ). iNOSis produced by several types of cell
transcription factor named nuclear factor B
and
(NF-B). NF- B is a transcription factor that is
is
the
key
mediator
of
several
immunological response. iNOS plays a role in
responsible
the release of NO plays a role in the
proinflammatory cytokines such as TNF-,
for
the
formation
of
several
pathogenesis of septic shock.
2
interleukin-1 (IL-1), IL-6, and IL-8 (Farris,
hours of treatment, venous blood sampling were
2005).
taken again to determine serum levels of NO in
Based on the above background, a problem is
both groups.
defined as whether giving vitamin C 3000mg
NO levels examinations conducted by
may reduce levels of nitric oxide (NO) in the
Griess method using a Cayman Chemical Nitrate
blood of severe burns.
/ nitrite assay.
For the safety of this procedure, the
drugs for the management of adverse drug
reaction
(unwanted
drug
reactions)
were
prepared and observation was done to the patient
MATERIAL AND METHOD
every hour in the Burn Unit. Patients were
This study is a randomized clinical trial.
The reached populations in this study are severe
burns patient post resuscitation Baxter admitted
to dr.Soetomo, Surabaya. Patients with severe
dropped out of this research if they were
incapable to follow the research due to their
basic disease, heart failure, unwanted drug
reactions or the wishes of the patient.
burns with multiple trauma, patients with HIV,
liver disorders, respiratory, heart and kidneys
were excluded from this study.
History
was
taken
and
physical
examinations were done. Laboratory tests
include complete blood count, BUN, serum
creatinine and Blood Gas Analysis were taken.
Then thesample were randomized.
RESULT
In this study, 12 severe burns patients
who matched the inclusion and exclusion criteria
were divided into 2 groups. Group 1 (K1) as the
control group and group 2 (K2) as the group
The study is divided into two groups.
Control group (K1) is the burn patients who
were given standard therapy of vitamin C
2x400mg / 24 hours for 72 hours, the treated
group (K2) is the burn patients were given
intravenous vitamin C 3000mg/24 hour for 72
hours.
receiving vitamin c 3000m, 6 patients each
group. 7 patients (58.3%) are male and 5
(41.7%) patients are women.
The mean age was 36.17 ± 12.99 years.
The mean age of group K1 was 39.2 ± 15.2 , the
youngest was 17 years old 61 years. The mean
age of group K2 is 33.2 ± 10.9 years, the
Peripheral
venous
blood
sample
(venouscubiti) must be taken as much as 3 cc in
both study groups for examination of serum NO
youngest was 20 and the oldest was 51 years.
The extent of burn in this study was 40.2
± 18.1 percent averages. In K1group the patients
levels before underwent the treatment. After 72
3
suffered 32.8 ± 16.6 percent of burnand 47.6 ±
30.00
17.7 percent in K2group. Data of patient
25.00
characteristics in both groups are shown in Table
20.00
serum
kreatinin
1.
15.00
BUN
Leukosit
10.00
pH darah
5.00
Table 1.Characteristics of Subject’s Study
0.00
Albumin
H1
Variables
Control n
=6
Vitamin
C, n = 6
H4
Price
p
Leucocytes Serum Creatinine BUN Blood acidity albumin
1.000
Sex
Man
3 (50,0)
Female
P value
4 (66,7)
0,01
0,85
0,21
0,14
0,63
Figure 2. Diagram characteristics leukocyte levels, sk, BUN, blood
3 (50,0)
2 (33,3)
Age
39.2 ± 15.2
33.2 ± 10.9
0.450
Extensive
burns (%)
32.8 ± 16.6
47.6 ± 17.7
0.166
pH, albumin group vitamin c (K2
Average levels of leukocytes, serum
creatinine, BUN and blood pH encountered no
Factors that may affect this study are
significant difference in the fourth day compared
shown in the picture below
to the first day in the control group (K1) (p>
0.05). While the levels of albumin in the control
14.00
Leukosit
group was (K1) significantly decreasing with p
12.00
10.00
= 0.04 (p <0.05).
serum
kreatinin
8.00
In the vitamin C group (K2),the amount
BUN
6.00
of leukocytes decreased significantly p = 0.01 (p
4.00
pH darah
<0.05) in the fourth day compared to the first
2.00
0.00
day. While the levels of serum creatinine, BUN,
Albumin
H1
H4
blood
pH
and
albumin
are
encountered
insignificant difference.
Leukocyte Serum Creatinine BUN Blood acidity Albumin
P value
0,13
1,0
0,61
0,86
Figure 1.Diagram Characteristics levels of leukocytes, SK, BUN,
blood pH, albumin control group (K1)
0,04
Prior to the statistical test, the normality
tests on the levels of Nitric Oxide (NO) were
madebefore and after treatment. Normality test
isused
to
determine
whether
clinical
or
laboratory parameter data distributed normally.
Normality test of levels of NO were doneusing
the Kolmogorov-Sminov techniques. The results
4
of normality test serum NO levels shown in the
7.0
tables below.
6.0
5.0
Table 2.Normality test results of serum levels of
NO
KelompokKontrol (K1)
Kelompok Vitamin C (K2)
Hari 1
Nilai
Hari 4
0,73
0,98
0,55
0,98
4.0
Kontrol
3.0
Delta
NO
0,91
0,77
2.0
1.0
0.0
H1
According to the table, serum levels of
H4
NO in the control group (K1) and vitamin c
group (K2), in the first and fourth day normally
distributed with p> 0.05.
By using a paired t test analysis to
Variables
Day 1
Day 4
value p
Control
4.7 ± 3.2
6.5 ± 3.3
0.21
(K1)
Figure 3.Diagram Analysis of the mean NO control group (K1).
analyze changes in the levels of Nitric Oxide
7.0
(NO) before and after treatment in each group, a
6.0
difference was found in the control group (K1).
5.0
Trend of increasing Nitric Oxide levels from 4.7
4.0
± 3.2 μM before treatment to 6 , 5 ± 3.3 μM
3.0
2.0
after treatment. However, this increase was
Vitamin C (K2)
1.0
statistically not significant (p = 0.21 (p> 0.05)).
0.0
H1
H4
At the Vitamin C 3000mg group,
decreased levels of Nitric Oxide (NO) were
found at 6.6 ± 3.1 μM levels before treatment to
3.8 ± 1.4 μM after treatment (p = 0.06 (p> 0.05).
For more details,see Figures 3 and 4.
Variabel
day 1
day 4
value p
Vitamin c (K2)
6,6±3,1
3,8±1,4
0,06
Figure 4.Diagram Analysis of the mean NO group vitamin c (K2).
8
6
4
H1
2
H4
Delta
0
Kontrol
Vitamin C 3000mg
-2
-4
Figure 5.Diagram Analysis of the results of the study control group
(K1) and the vitamin c (K2)
5
Independent t test was done to analyze
The
release
of
pro-inflammatory
the mean difference between the control group
cytokines (TNF-α, IL-1 and IL-6) is an
(K1) with Vitamin C 3000mg group. The control
important mechanism in the regulation of the
group gained an increase in the average levels of
acute phase response to burns. TNF-α is a
Nitric Oxide (NO) as much as 1.8 ± 3.1 μM
triggering cytokine that induce a cascade of
levels from 4.7 ± 3.2 μM before treatment to 6.5
secondary cytokines and humoral factors then
± 3.3 μM after treatment. The vitamin C 3000mg
lead to systemic and local sequele. And then
obtained a decrease of -2.6 ± 2.7 μM from 3.1 ±
TNF-α is involved in the development of
6.6 μM levels before treatment to 3.8 ± 1.4 μM
conditions such as shock associated with burns
after treatment. Analysis of NO difference
and sepsis (Cakir B, 2004).
(Delta NO) showed a significant reduction in the
Nitric oxide is a biological molecule
value of p = 0.02 (p <0.05) in the comparison
produced by different cell types, have a good
between the NO on the vitamin C (K2) with NO
and bad effect as well to the blood vessels and
in the control group (K1).
cellular level. NO is an important key to the
pathogenesis of sepsis. INOS activation would
lead to the formation of large numbers NO and
showed that L-arginine is available in sufficient
quantities (Chen 1999).
DISCUSSION
Schorah C et al reported a study
Local and systemic changes are caused
conducted on patients admitted in the ICU that
by inflammatory mediators. Severe burns also
showed lower levels of total vitamin C, ascorbic
cause depression of cellular andhumoral immune
acid and dehydroascorbic acid than in patients
response and phagocytes aspects of blood-borne
with gastritis, diabetes, and healthy people.
macrophages.
Burns
initiate
systemic
Long et al. found that patient’s ascorbic
inflammatory reaction caused by toxins and
acid plasma levels on parenteral dose of 300 mg
burns oxygen radicals and eventually lead to
/ day of ascorbic acid for 2 days is unresponsive.
peroxidation. Reactive oxygen metabolites cause
Plasma levels began to rise at 1000 mg / day in 2
destruction or damage to the cell membrane by
days but still below normal levels, it takes 3
lipid peroxidation. The relationship between the
days or more after the parenteral dose 3000 mg /
number of products of oxidative metabolism and
day to achieve normal plasma levels.
free radical scavenger naturally determine the
Transcription factor NF-кB has a crucial
result of local and systemic tissue damage, also
role in the inflammatory process. NF-кB is a
organ failure subsequent to the burn (J Horton,
transcription factor that triggers the production
2003).
of cytokines. Administering of LPS can activates
6
the NF-кB which increases the production of
illness, as well as the decrease in the ratio of
inflammatory mediators such as IL-8, TNF-α,
ascorbic
intercellular
concentration of Vitamin C levels in patients
adhesion
molecule
(ICAM)
dancyclooxygenase-2.
acid
to
dehidroascorbic.
Low
with sepsis noted that subjects likely susceptible
Vitamin C as an anti-inflammatory is
to oxidant stress.
also able to suppress the activation of the
In this study, the result of vitamin c
transcription factor nuclear factor  B (NF- B)
3000mg
and inhibit tumor necrosis factor  (TNF-).
decreasing of serum NO levels on day four
NF- B is a transcription factor that is
compared
responsible
decreasing
for
the
formation
of
several
administration
showedinsignificant
to day one. This insignificant
might
be
because
the
drug
proinflammatory cytokines such as TNF-,
administration period is too short. But there is a
interleukin-1 (IL-1), IL-6, and IL-8 (Farris,
significant decrease in the levels of NO in the
2005).
fourth day of from the first day (delta NO) in the
Observations showed that leukocyte
levels of the group given 3000mg of vitamin c
group given a vitamin c 3000mg (K2) compared
with the group without vitamin c (K1).
Antioxidants are the body's protective
decline significantly in the fourth day compared
system against free radical activity generated
to the first day.
As an antioxidant, vitamin C can react
both endogenous and exogenous which is owned
and
by every normal cells. Antioxidants are defined
phase,
as components that can protect itself against
converting free radicals to more inertform.
oxidation process that can turn it into free
Arachidonic acid cascade, which is activated by
radicalsdespite the very small amount when
ROS will be cut so that the inflammatory
compared to other components.
with
ROS
produced
macrophages
in
the
by
neutrophils
inflammatory
anti-
Giving high doses of vitamin C will
inflammatory effects allegedly associated with
cause diarrhea, kidney stones and kidney
vitamin C as an antioxidant nature (Lima, 2009).
dysfunction. In this study there was no
reaction
will
stop.
Therefore,
the
Galley et al. discovered in his study of
significant
increase
of
BUN
and
serum
vitamin C concentration of total pre-infusion in
creatininlevels in the group received 3000 mg of
septic patients is lower than healthy subjects.
vitamin C in the fourth day compared to the first
This study indicated that patients in sepsis
day.
syndrome had a quite muchlower total vitamin C
This study has several limitations such
than normal controls, and it was consistent with
as NO examination cannot be performed in dr.
previous studies that found low levels of
Soetomo hospital, and it is difficult to achieve
ascorbic acid in the group of patients at critical
thenumberof sample of new severe burns
7
patients which matched the inclusion criteria in
determined time.
BogdanC,2001.
Nitric
oxide
and
the
immuneresponse.Nature immunology 2(10), pp.
907-916.
Cakir B, Yegen B, 2004. Systemic response to burn
injury. Turk J Med Sci, pp. 215 – 226.
Chen LW, Hsu CM, Cha MC,1999,. Changes in gut
mucosal nitric oxide synthase (NOS) activity
after thermal injury and its relation with barrier
failure. Shock, 11,pp 104-110.
CONCLUSION
There
is
a
statistically
significant
decrease of levels NO (delta NO) between the
group that was given intravenous vitamin c 3000
mg/24 hours for 72 hours with the control group
which is given intravenous vitamin c 2x400mg
for 72 hours in patients with severe burns.
Chen X, Soejima K, 2004. Effect of Early Excision on
Changes in Plasma Nitric Oxide and Endothelin1 Level After Burn Injury : an Experimental
Study in Rats. Burn, 30. pp. 793-797.
Colven RM, Pinnell SR, 1996. Topical vitamin C in
aging.Clinics in dermatology. New York:
Elsevier Inc., 14, pp.227-234.
Da Silva M, Filipe H, 1998. Nitric oxide and human
thermal injury short term outcome, J Burn,24,
pp. 207-212.
ADVICE
There needs to be studies with a larger
sample, a longer period of time accompanied by
measurement of total vitamin C plasma levels
earlier in severe burn patients.
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