EL-MINIA MED., BULL., VOL. 19, NO. 1, JAN., 2008
Osman et al
PLASMA MITOCHONDRIAL DNA
CONCENTRATIONS AFTER TRAUMA
By
Ashraf M. M. Osman*, MD; Tohamy AT, MD;** , and Wael Talat, MD***
Departments of *Clinical Pathology, **General surgery, ***Neurology ,
Minia Faculty of medicine
ABSTRACT:
Background: DNA concentrations increase in the circulation of patients after trauma
and may have prognostic value. The aim of this study is to investigate the temporary
changes in plasma DNA concentrations in patients after trauma.
Patients and methods: Serial blood samples were taken from twenty patients with
blunt trauma within the first 3 h of admission to General Surgery Department and on
daily intervals for two days.
Results plasma DNA was increased within 30 min of injury and was significantly
higher in patients with severe injury and in patients who developed organ failure and
it correlates with C-reactive proteins and serum albumin.
Conclusions: Plasma DNA concentrations increase early after injury and are higher in
patients with severe injuries and in those who develop organ failure. Increased plasma
DNA persists for days after injuries, especially in patients with multiple organ
dysfunction syndrome. So it was found to be useful in posttraumatic prognosis.
KEY WORDS:
DNA
Blunt trauma
Organ Failure
genome, Mitochondrial DNA concentration in plasma increases in patients
in the first few hours after trauma,
which correlates with injury severity,
and it predicts late posttraumatic
complications such as organ failure,
acute lung injury, and death; and that it
may be useful, especially when
combined with other predictors in a
prediction guideline4.
INTRODUCTION:
Trauma is characterized by
tissue necrosis and cell death whether
as a result of immediate direct injury or
as a delayed inflammatory reaction1.
Increases in plasma DNA occur in a
variety of conditions associated with
cell death, including cancer, pregnancy
and stroke. Although the mechanisms
by which nucleic acids are released
into the circulation are unknown, it is
likely that cell death is a major factor2.
These studies involved only one
blood sample taken from each patient,
and it was evident that there was
considerable variation among patients.
Such variability may be explained by
rapid changes in the balance of
production and clearance of DNA in
the plasma. Fetal DNA in maternal
plasma has been shown to have rapid
clearance kinetics, and this may be
typical of other forms of DNA5.
Significant
increases
in
circulating DNA in the plasma of
trauma patients have been reported and
found to be useful in posttraumatic
prognosis3.
Mitochondria,
intracellular
powerhouses generating the cellular
energy source ATP, have their own
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EL-MINIA MED., BULL., VOL. 19, NO. 1, JAN., 2008
In the present study, it is
investigated the concentration–time
relationship of plasma DNA (using the
ß-globin gene as a marker) in the initial
few hours of injury and after admission
in the General Surgery, Minia
University Hospital.
Osman et al
2- Complete liver
and renal
functions on automated clinical
chemistry Dimension ES.
3- High sensitive
C-Reactive
proteins.
4- mt DNA level
Blood sampling:
A maximum of three separate
blood samples were withdrawn from
each patient in this study. 3 ml
collected of peripheral venous blood
from each patient into heparin
containing tubes after the patients were
admitted to the resuscitation room. The
median time between injury and the
first blood collection was within range,
30–60 min, and on daily intervals for
two blood samples
SUBJECTS AND METHODS:
This study was conducted
between August 2007 and December
2007 in the Departments of General
Surgery and Clinical Pathology, Minia
University Hospital. The study was
investigated early changes within the
first 3 h after injury and on daily
intervals for two days in twenty
patients who had sustained an acute
blunt traumatic injury and had been
admitted to the resuscitation room at
the Departments of General Surgery
and ten apparently healthy persons as
control group. Inclusion criteria
included time from injury to admission
of <4 h and age >13 years. Exclusion
criteria were pregnancy, drowning,
hanging, thermal injury, hypothermia,
and acute drug overdose. It involved
taking blood
samples at daily
intervals
to observe changes in
plasma DNA concentrations in severely injured patients.
preparation of plasma DNA
Blood samples were centrifuged at 1500g for 10 min, and plasma
was then transferred into plain
polypropylene tubes and stored at 80°C. DNA was extracted from 200µL plasma samples with use of a
QIAamp Blood Kit (Qiagen) according
to the "blood and body fluid protocol"
as recommended by the manufacturer.
Plasma DNA was measured by
quantitative PCR assay for the ß-globin
gene, which is present in all nucleated
cells of the body. The ß-globin PCR
system consists of the amplification
primers ß-globin-354F (5'-GTG CAC
CTG ACT CCT GAG GAG A-3') and
ß-globin-455R (5'-CCT TGA TAC
CAA CCT GCC CAG-3'). The PCR
probe contained a 3'-blocking phosphate group to prevent probe extension
during PCR (Lo YMD et al., 1998). It
was applied to serial dilutions of
human genomic DNA, then detection
through gel electrophoresis 1.2%, The
ethidium bromide stained gel was
examined under ultraviolet light. The
size of the bands was assessed by
direct comparison with standard weight
Examination of different groups:
* General examination: Vital
data: pulse, blood pressure, temperature, respiratory rate. Abdominal
and chest examination: detection
thetype of trauma and severity of
case presentation.
* Neurological examination:
detection
motor
or sensory
affection and also degree of
conscious.
* Laboratory investigations:
1- Complete blood count on
automated cell count, sysmex NE.
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EL-MINIA MED., BULL., VOL. 19, NO. 1, JAN., 2008
markers6. The relative intensity of the
study patients versus control group.
Osman et al
mtDNA
concentrations revealed
positive correlations. Moreover, the
median plasma mtDNA concentration
in trauma patients who later died was
higher than that of the survivors.Within
the first hour, the overall mean DNA
concentration was three fold higher in
patients with severe injury.
RESULTS:
Twenty patients with acute blunt
trauma (mean age, 38 years; 13 males
and7 females) were studied.The
median concentrations of DNA were
both significantly higher in trauma
patients than in the controls.
The mean plasma DNA
concentration on the day of injury (day
0) was increased in trauma patients
compared with healthy controls.
Comparison of the severity of the
injury with the corresponding plasma
Table 1: mt DNA , CRP & Albumin in patients group and control group.
Mt DNA
mmol/ml
CRP
mg/dl
Albumin
g/dl
Control
group
1.8-2.1
Patients
group1st day
2.4-5.3
Patients
group2nd day
3.9-6.7
Patients group
3rd day
2.6-5.1
0-4.3
16-48
40-96
40-85
4.3-5.1
3.9-4.8
3.6-4.3
3.6-4.1
Table 2: Corrletions between mt DNA , CRP & Albumin in patients group and
control group.
Mt DNA group1st day
Mt DNA
group2ndday
Patients group 3rd day g/dl
CRP
Albumin
0.3
NS
0.05
S
0.02
S
-0.04
NS
0.28
NS
0.13
NS
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EL-MINIA MED., BULL., VOL. 19, NO. 1, JAN., 2008
Osman et al
patients with severe injuries and in
those who develop organ failure. It
significantly correlates with C-reactive
protein level while not with serum
albumin. These concentrations remain
increased for days after injury. These
findings may be used for risk
stratification and monitoring posttraumatic disease processes.
DISCUSSION:
Plasma DNA concentrations
increase early after injury and are
higher in patients with severe injuries
and in those who develop organ failure.
Increased plasma DNA persists for
days after injuries, especially in
patients
with
multiple
organ
dysfunction syndrome7. High concentrations of plasma DNA observed in
severely or multiply injured patients
suggests that extracellular DNA may
originate from damaged tissues, i.e.,
necrosis. Apoptosis is a complex
process highly regulated by many
genes and may take several hours to
develop.
The high plasma DNA
concentrations detected in this study
occur as early as 30 min after trauma
Ngek et al., 2003. Lo et al., 2000
studies had also reported that
apoptosis,
at
least
in
polymorphonuclear leukocytes, is
suppressed
in
patients
with
posttraumatic complications such as
the adult respiratory dysfunction
syndrome8. This suppression may be
attributable to the presence of high
concentrations
of
inflammatory
mediators in the circulation. Impaired
clearance is another possible reason for
the increase in cell-free DNA. In
healthy pregnant women, plasma DNA
after delivery has an extremely short
half-life in the circulation . However,
after trauma, organs responsible for
elimination might be damaged as a
consequence of ongoing systemic
inflammation9. A previous study
indicated that the sequential hourly and
daily changes in plasma ß-globin DNA
concentrations after trauma were
different between mildly and severely
injured patients and between patients
with and without organ failure The
differences may also be useful for
monitoring the progress of post-trauma
complications10. In conclusion, plasma
DNA concentrations are increased
early after injury and are higher in
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LYS, Joynt GM, Lo YMD: Plasma
Mitochondrial DNA Concentrations
after
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Clin
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2004;50:213-216.
2. Keel M, Ecknauer E, Stocker
R, Ungethum U, Steckholzer U,
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with chest trauma. J Trauma 1996;
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3. Ngek O, Tsui NBY, Lau TK,
Leung TN, Chiu RWK, Panesar NS:
Messenger RNA of placental origin is
readily detectable in maternal plasma.
Proc Natl Acad Sci U S A
2003;100:4748-4753
4. Park HK, Kim SK, Kim MS,
Cho EY and Lee JH: Fetal and early
postnatal proteins malnutrition cause
long term and early postnatal proteins
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in rat liver and muscle mitochondria. J
Nutr , 2003; 133:3085=3090.
5. Valdez R Athens M, Thompson
G, Bradshaw B and stern M: birth
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bi-ethnic population in the USA.
Diabetologia 1994; 37: 624-631,.
6. Chiu RWK, Chan LYS, Lam
NYL, Tsui NBY, Ng EKO, Rainer TH:
Quantitative analysis of circulating
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Chem 2003;49:719-726.
7. Lo YMD, Tein MSC, Lau TK,
Haines CJ, Leung TN, Poon PMK:
Quantitative analysis of fetal DNA in
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‫‪plasma‬‬
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‫‪serum:‬‬
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‫‪1998;62:768-775.‬‬
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‫‪LYS, Hjelm NM, Cocks RA:. Plasma‬‬
‫‪DNA as a prognostic marker in trauma‬‬
‫‪patients. Clin Chem 2000;46:319-323.‬‬
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‫‪Shin CS: Changes in mitochondrial‬‬
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‫تجمعات الشريط الوراثى الميتوكوندري فى البالزما‬
‫بعد التعرض لالصابات التصادمية‬
‫اشرف عثمان* ‪ ,‬تهامى عبدهللا* ‪ ,‬وائل طلعت***‬
‫اقسام *التحاليل الطبية ‪** ,‬الجراحة العامة ‪*** ,‬العصبية والنفسية‬
‫كلية طب المنيا‬
‫تزيد تجمعات ‪ DNA‬بعد االصابات التصادمية ‪ ,‬وهذا ربما يكون له قيمة تنبؤية ‪ .‬إن هدف هذه‬
‫الدراسة أن تتحرى التغييرات المؤقتة في تجمعَّات بالزما ‪ DNA‬في المرضى بعد االصابات‬
‫التصادمية‪.‬‬
‫المرضى وطريقة البحث‪ :‬اشتملت هذه الدراسة على ‪ 20‬مريض تم ادخالهم قسم الجراحة‬
‫العامة بعد تعرضهم الصابات تصادمية ‪ ,‬حيث اخذت لجميع المرضى عينات دم متسلسلة‬
‫ضمن أول ‪ 3‬ساعات من التعرض لالصابة ثم عينة يومية لليومين التاليين‪.‬‬
‫نتائج البحث ‪ :‬وجد ان تجمعات ‪ DNA‬فى البالزما تزيد فى خالل ‪ 30‬دقيقة من التعرض‬
‫لالصابة ‪ ,‬وكان عالى جدا في المرضى الذين تعرضوا الصابة شديدة ‪ ,‬وفي المرضى الذين‬
‫سائت حالتهم وتطورت الى فشل فى وظائف االعضاء وهو يرتبط مع ‪ C‬بروتين وزالل الدم‪.‬‬
‫اإلستنتاجات‪ :‬اثبتت هذه الدراسة ان تجمعَّات ‪ DNA‬تزيد فى البالزما مبكرا بعد التعرض‬
‫لالصابة التصادمية الرادة ‪ ,‬وأعلى في المرضى باإلصابات الشديدة وفي أولئك الذين تتطور‬
‫حالتهم وتسوء الى فشل فى وظائف االعضاء‪ .‬ويستمر ‪ DNA‬المتزايد فى البالزما لعدة أيام‬
‫بعد التعرض لإلصابات الرادة‪ ،‬خصوصا في المرضى الذين سائت حالتهم الي االصابة‬
‫بمتالزمة عطل االعضاء المتعددة‪ .‬لذلك قد يكون من المفيد متابع ال ‪ DNA‬في الدم بعد‬
‫التعرض لالصابات ‪ ,‬حيث انه سوف يساعد فى التنبوء فى تشخيص المضاعفات ومحاولة‬
‫تجنبها‪.‬‬
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