1. C o m b a t C a s u a l t y C a r e
P
R O T E C T
PRO
J E C T - S U S T
AIN
T
INSTITU
T E
O F S U R G I C A L R E
S
EARCH
Wavelength (nm)
200 300 400 500
Absorbance(mAb)
-100
0
100
200
300
400
500
Wavelength (nm)
200 300 400 500
Absorbance(mAb)
-100
0
100
200
300
400
500
600
Wavelength (nm)
200 300 400 500
Absorbance(mAb)
-100
0
100
200
300
400
500
Bufodienolide
Anthraniloyl- Bufodienolide
Bufalin
Cinobufagin
Resibufogenin
Time (min)
8 10 12 14 16 18 20
Absorbance(mAb)
0
50
100
150
200
250
300
0.2µg
0.5µg
1.0µg
2.0µg
4.0µg
Bufalin
Cinobufagin
λ=355nm
Resibufogenin
Injected into the HPLC
µg/ml
0 2 4 6 8 10
Absorbance(355nm)
0
20
40
60
80
100
120
Y=mx+b
m=10.9
b=10.5
R2=0.992
O
O
O
O
H
H
H
O
NH
CH3
µg/ml
0 2 4 6 8 10
Absorbance(355nm)
0
20
40
60
80
100
120
140
Y=mx+b
m=13.5
b=10.3
R2=0.994
O
O
O
O
O
O
H
H
H
O
NH
CH3
µg/ml
0 2 4 6 8 10Absorbance(355nm)
0
20
40
60
80
100
120
140
160
180
200
Y=mx+b
m=19.3
b=24.3
R2=0.996
H
OH
H
H
O
O
O
O
NH
CH3
Bufodienolides were extracted by the following
process: 1mL of plasma was spiked with 4, 2,
1, 0.5, and 0.2 µg of bufalin, cinobufagin, and
resibufogenin. 10mL of Methylene Chloride
was added and centrifuged at 2000 rpm. The
solution phase-separated, and the bottom
phase (Methylene Chloride) was removed and
air dried. This portion was then re-dissolved in
10% Acetonitrile (MeCN) and extraction
continued using a HLB OASIS Column (Waters
Inc.). The column was washed in 2ml of MeCN
and 1ml of H2O. The sample was added to the
column and washed in 1ml 10% MeCN / 2%
Formic Acid and 1ml 10% MeCN. The sample
was eluted in 3ml of MeCN and dried under air.
Bufodienolides were conjugated to a
fluorescent probe: 400ul of N-methylisatoic
anhydride (NMIA) solution was added to the
sample and incubated at 90°C for 24 hours.
The NMIA solution contained 0.50 ml of 0.1M
NMIA, dimethyl aminopyridine, 4-pyrrolidino
pyridine, and 3.85ml MeCN. The NMIA
chemically combined with the bufodienolides to
create an anthraniloyl conjugate (Figure 1).
The purpose of this study is to develop a
method to measure plasma levels
bufodienolides in hemorrhagic shock. . The
method will begin by extracting the
bufodienolides out of plasma, conjugating the
bufodienolides to a fluorescent probe, and then
measuring the bufodienolide-fluorescent
conjugate on High Pressure Liquid
Chromatography (HPLC).
1) Bufalin, Cinobufagin, and Resibufogenin
were successfully extracted from pig
plasma.
2) We were also able to successfully
conjugate NMIA to these bufodienolides
(Figure 2).
3) The bufodienolide-conjugates were then
separated and measured on HPLC (Figure
3).
4) Varying concentrations of the
bufodienolides led to a dose-dependent
rise in the peak height on the HPLC
(Figure 3).
5) Standard curves were generated for
bufalin, cinobufagin, and resibufogenin
that fit a straight line with a R2
= 0.99
(Figures 4 – 6).
1) We have now developed a method that can
extract and measure bufodienolides in
plasma.
2) This method can be extended to measure
bufodienolides in plasma of patients with
hemorrhagic or other forms of shock.
.
The anthraniloyl conjugates were measured
on HPLC: A Beckman Coulter HPLC with
fluorescent and UV-Visible Multi-array
detectors was used. The HPLC gradient was
set at 85-100% MeCN over five minutes. The
conjugates were separated and peak heights
were measured at λ = 355 nm (UV) and
fluorescence (la = 355 nm, le = 435 nm)
(Figure 3).
The use of Army medical and/or other Army records in the preparation of this material is acknowledged, but is not to be construed as implying official Department of the Army approval of the conclusions presented.
NEW METHOD FOR MEASURING ANTHRANILOYL-BUFODIENOLIDES IN
PLASMA BY HPLC
David A. Ocon, Cassandra Moreno and Daniel N. Darlington, PhD
The Pittsburg Tissue Engineering Institute and
United States Army Institute for Surgical Research, Fort Sam Houston, TX 78234-6315
Results
Introduction
Objectives
Methods
Conclusions
References
A critical issue for both the military and civilian
population is hemorrhagic shock. About 85% of
potentially survivable deaths in the war in Iraq
were due to hemorrhage (1). Recent studies
show that normal restoration of plasma volume
fails after inhibition of Na/K ATPase and turn a
normally non-lethal hemorrhage into a lethal
one. Bufodienolides, a large family of powerful
Na/K ATPase inhibitors, originally isolated from
amphibia, have recently been found in human,
mouse, and rat plasma (2-4).
Figure 1.
Conjugation of NMIA to the Bufodienolides
Figure 4.
Bufalin-Conjugate Standard Curve
Figure 5.
Cinobufagin-conjugate standard curve
Figure 3.
Dose-dependent changes in peak height
Figure 6.
Resibufogenin-conjugate standard curve
Figure 2.
Spectral Shift after conjugation
1) Kelly, J.F., et al. Injury Severity and Causes of Death
From Operation Iraqi Freedom and Operation Enduring
Freedom: 2003-2004 Versus 2006. J Trauma. 2008;64:S21-
S27.
2) Bagrov, A. Y., R. I. Dmitrieva, et al. "Endogenous
marinobufagenin-like immunoreactive substance. A possible
endogenous Na, K-ATPase inhibitor with vasoconstrictor
activity." Am J Hypertens 1996:9(10 Pt 1): 982-90.
3) Bagrov, A. Y., O. V. Fedorova, et al. "Endogenous
marinobufagenin-like immunoreactive factor and Na+, K+
ATPase inhibition during voluntary hypoventilation."
Hypertension 1995:26(5): 781-8.
4) Butler, V. P., Jr., J. F. Morris, et al. "Heterogeneity and
lability of endogenous digitalis-like substances in the plasma
of the toad, Bufo marinus." Am J Physiol 1996:271(2 Pt 2):
R325-32.