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Copper-induced oxidation of epinephrine: protective effect ofDD-DAHK,

a synthetic analogue of the high affinity copper

binding site of human albumin

Angela Roberts,a David Bar-Or,a,b,* James V. Winkler,b and Leonard T. Raela,b

a Department of Trauma Research, Swedish Medical Center, 501 E. Hampden Avenue, Englewood, CO 80110, USAb DMI BioSciences Inc., 3601 South Clarkson Street #420, Englewood, CO 80110-3944, USA

Received 2 April 2003

Abstract

Epinephrine is known to be rapidly oxidized during sepsis. Ischemia and acidosis, which often accompany sepsis, are associated

with the release of weakly bound cupric ions from plasma proteins. We investigated whether copper promotes oxidation of epi-

nephrine at both physiological and acidic pH and whether DD-AspDD-AlaDD-HisDD-Lys (DD-DAHK), a human albumin (HSA)

N-terminus synthetic peptide with a high affinity for cupric ions, attenuates this oxidation. Epinephrine alone [100 lM] or with

CuCl2 [10 lM], and with CuCl2 [10 lM] and DD-DAHK [20 lM] at pH 7.4, 7.0, 6.5, and 6.0 were incubated for 1 h at 37 C. Epi-

nephrine oxidation was measured by the spectrophotometric quantification of its oxidation product, adrenochrome. We found that

adrenochrome increased, suggesting copper-induced oxidation of epinephrine. At pH 7.4, 7.0, 6.5, and 6.0, adrenochrome increased

by 47%, 53%, 24%, and 6% above baseline, respectively. DD-DAHK attenuated the copper-induced oxidation of epinephrine to

baseline levels. These in vitro results indicate that copper-induced epinephrine oxidation is greatest at the physiological pH 7.4 as

well as in severe acidosis, pH 7.0, and that DD-DAHK completely inhibits this oxidation.

2003 Elsevier Science (USA). All rights reserved.

Keywords: Epinephrine; Adrenaline; Sepsis; Ischemia; Acidosis; Copper;DD-AspDD-AlaDD-HisDD-Lys; DAHK; Human albumin

Sepsis is the most common cause of mortality in the

ICU, with a mortality rate of 3040% in all sepsis pa-

tients and reaching as high as 80% in patients with septic

shock [1]. Despite recent advances in the treatment of

sepsis, mortality remains very high [2,3]. Sepsis is char-

acterized by a decline in blood pressure and a resistance

to vasoconstrictor therapy [4,5]. Epinephrine is rapidly

oxidized during sepsis to adrenochrome [6]. The rapidoxidation of epinephrine prevents its action as a vaso-

constrictor and adrenochrome itself has been shown to

be cardiotoxic [710].

Copper has been reported to promote the in vitro

oxidation of catecholamines, including epinephrine [11

13]. General or localized acidosis, which occurs in sepsis

and other illnesses [14,15], results in the release of copper

from ceruloplasmin and other proteins in the circulation

[1618]. Previous studies in our laboratory have dem-

onstrated that copper inhibits the anticoagulant activity

of activated protein C which is used to treat sepsis [19]. In

the present study, we sought to determine whether cop-

per-induced oxidation of epinephrine is pH dependent in

physiological ranges and, if so, whether the introduction

of the human albumin N-terminus, DD-AspDD-AlaDD-

HisDD-Lys (DD-DAHK), which has a high affinity for

cupric ions, will protect epinephrine from oxidation. Theresults of this in vitro experiment showed that after

60 min of incubation, the oxidation of epinephrine was

increased twofold when copper was present at pH 7.4

and 7.0 but had little effect at pH 6.0 and 6.5. However,

at all pH tested, the addition of DD-DAHK inhibited the

oxidation of epinephrine to levels at or below baseline.

Experimental procedures

Materials. The human albumin N-terminus analogue, DD-DAHK,

was synthesized by Bowman Research (Newport, Wales, UK).

Biochemical and Biophysical Research Communications 304 (2003) 755757

www.elsevier.com/locate/ybbrc

BBRC

* Corresponding author. Fax: 1-303-788-4064.

E-mail address: dbaror@dmibio.com (D. Bar-Or).

0006-291X/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(03)00667-3

http://mail%20to:%20dbaror@dmibio.com/http://mail%20to:%20dbaror@dmibio.com/

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(Englewood, CO). We thank Richard Shimonkevitz, PhD, and Greg-

ory Thomas, BS, for their technical assistance in this research.

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