1. NEW METHOD FOR MEASURING ANTHRANILOYL-BUFODIENOLIDES IN
PLASMA BY HPLC
David A. Ocon*1
, Cassandra Moreno *2
, and Daniel N. Darlington, PhD*2
.
1*
Summer Intern, Pittsburgh Tissue Engineering Initiative.
2*
US Army Institute of Surgical Research, Fort Sam Houston, Texas.
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). The purpose of this study is to develop a method to measure plasma
levels of 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).
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 µl 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.
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 (λa = 355 nm, λe = 435 nm). Standard curves were
generated for bufalin, cinobufagin, and resibufogenin that fit a straight line with a R2
= 0.99.
Bufalin, Cinobufagin, and Resibufogenin were successfully extracted from pig plasma. We were
also able to successfully conjugate NMIA to these bufodienolides. The bufodienolide-conjugates
were then separated and measured on HPLC. Varying concentrations of the bufodienolides led to
a dose-dependent rise in the peak height on the HPLC. Standard curves were developed for each
bufodienolide. These standard curves should allow us to accurately measure bufodienolides in
mammalian plasma.
We have now developed a method that can extract and measure bufodienolides in plasma. This
method can be extended to measure bufodienolides in plasma of patients with hemorrhagic or
other forms of shock.

2. REFERENCES:
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(10bPt 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.