BBRC

Biochemical and Biophysical Research Communications 304 (2003) 755757 www.elsevier.com/locate/ybbrc

Copper-induced oxidation of epinephrine: protective eect of D -DAHK, a synthetic analogue of the high anity 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, USA b 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 epinephrine at both physiological and acidic pH and whether D -AspD -AlaD -HisD -Lys (D -DAHK), a human albumin (HSA) N-terminus synthetic peptide with a high anity for cupric ions, attenuates this oxidation. Epinephrine alone [100 lM] or with CuCl2 [10 lM], and with CuCl2 [10 lM] and D -DAHK [20 lM] at pH 7.4, 7.0, 6.5, and 6.0 were incubated for 1 h at 37 C. Epinephrine oxidation was measured by the spectrophotometric quantication 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. D -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 D -DAHK completely inhibits this oxidation. 2003 Elsevier Science (USA). All rights reserved.
Keywords: Epinephrine; Adrenaline; Sepsis; Ischemia; Acidosis; Copper;
D -AspD -AlaD -HisD -Lys;

DAHK; Human albumin

Sepsis is the most common cause of mortality in the ICU, with a mortality rate of 3040% in all sepsis patients 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 characterized by a decline in blood pressure and a resistance to vasoconstrictor therapy [4,5]. Epinephrine is rapidly oxidized during sepsis to adrenochrome [6]. The rapid oxidation of epinephrine prevents its action as a vasoconstrictor 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* Corresponding author. Fax: 1-303-788-4064. E-mail address: dbaror@dmibio.com (D. Bar-Or).

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 copper-induced oxidation of epinephrine is pH dependent in physiological ranges and, if so, whether the introduction of the human albumin N-terminus, D -AspD -AlaD HisD -Lys (D -DAHK), which has a high anity for cupric ions, will protect epinephrine from oxidation. The results 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 eect at pH 6.0 and 6.5. However, at all pH tested, the addition of D -DAHK inhibited the oxidation of epinephrine to levels at or below baseline.

Experimental procedures
Materials. The human albumin N-terminus analogue, D -DAHK, was synthesized by Bowman Research (Newport, Wales, UK).

0006-291X/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00667-3

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A. Roberts et al. / Biochemical and Biophysical Research Communications 304 (2003) 755757

Epinephrine, adrenochrome, copper chloride, and all other chemicals were obtained from Sigma (St. Louis, MO). Adrenochrome standard curve. Solutions of adrenochrome were prepared in 10 mM KH2 PO4 buer (pHs 7.4, 7.0, 6.5, and 6.0) at the following concentrations: 100, 80, 60, 40, and 20 lM (n 2 for each), incubated for 60 min at 37 C, and read on a UVvisible recording spectrophotometer (Shimadzu Model UV160U) at 280 nm (7) against blanks containing only 10 mM KH2 PO4 buers. Epinephrine oxidation assay solutions. Solutions of CuCl2 , D -DAHK, and epinephrine were prepared in 10 mM KH2 PO4 buer at pH 7.4, 7.0, 6.5, and 6.0. Epinephrine (100 lM) was added to the following solutions at each pH: (a) 10 mM KH2 PO4 buer alone, (b) 10 CuCl2 , and (c) 10 lM CuCl2 plus 20 lM D -DAHK (n 3 for each). Samples were incubated for 60 min at 37 C and read on a UVvisible recording spectrophotometer (Shimadzu Model UV160U) at 280 nm (7) against blanks containing only 10 mM KH2 PO4 buers. Statistical analysis. Data were compared using paired, two-tailed Students t test with a computer program (Excel 2000, Microsoft, Redmond, WA) and signicance is reported when p < 0:05.

Fig. 1. D -DAHK inhibition of copper-induced epinephrine oxidation to adrenochrome.

Results and discussion Copper promotion of epinephrine oxidation is pH dependent There was no signicant variance across the range of pHs studied in the amount of spontaneous epinephrine oxidized in 1 h (n 3, p > 0:26). After 60 min, epinephrine alone with KH2 PO4 buer was oxidized to produce 23.526.7 lM of adrenochrome for all pH levels. The addition of copper increased the oxidation to adrenochrome at pH 7.4 and 7.0 to 53:0 lM 3:3 (p < 0:05) and 60:8 lM 6:8 (p < 0:05), respectively, a greater than twofold increase in epinephrine oxidation. At pH 6.5, copper promoted minimal epinephrine oxidation to an adrenochrome concentration of 32.6 lM only. No eect by copper was seen at pH 6.0. Although copper has been shown to promote epinephrine oxidation [1113], our results support the conclusion that copper does not signicantly induce epinephrine oxidation at pH levels below 7.0. This may be because acidic conditions are less conducive to oxidation. However, normal physiological pH (7.4) and the acidic pH (7.0), which may occur in patients with general or localized acidotic conditions [23], appear optimal for copper-catalyzed oxidation of epinephrine. Plasma proteins closely regulate copper under normal conditions. Acidic conditions are associated with the release of cupric ions from ceruloplasmin and other proteins [20]. Increased oxygen requirements, with impaired oxygen extraction and maldistribution of blood ow, create ischemia and acidosis in early sepsis and release free cupric ions [16,21,22]. Copper-induced deactivation of epinephrine may be a contributory factor to the pathogenesis of septic shock [6] and the formation of adrenochromes may contribute to cardiotoxicity [710] (see Fig. 1).

Results presented here demonstrate that at physiological and acidic pH free copper may accelerate epinephrine oxidation to adrenochrome. Acidosis in sepsis results in the release of loosely bound copper into the circulation at the conditions optimal for copper to act as an oxidizing agent for epinephrine, thereby diminishing endogenous supply of epinephrine and undermining the therapeutic ecacy of administered epinephrine. Thus, specic copper chelation could inhibit this accelerated epinephrine oxidation and may be benecial for the treatment of sepsis. In the present study, the N-terminal albumin analogue, D -DAHK, completely inhibited copper-induced oxidation of epinephrine at pH between 6.0 and 7.4 (p < 0:05). These results indicate that D DAHK could have therapeutic potential in conditions associated with cupric ion release by sequestering free copper and minimizing the oxidative deactivation of epinephrine. Inhibiting the degradation of endogenous epinephrine may reduce requirements for exogenous catecholamine administration and, thereby, reduce the formation of toxic catecholamine oxidation products. In addition to these in vitro results, other reports have shown that copper catalyzes the production of ROS, including the superoxide and hydroxyl radicals, [24]. ROS are known to catalyze the deactivation of catecholamines and are produced in sepsis [6,25,26]. We have previously reported that D -DAHK blocks ROS production in vitro [24]. Therefore, D -DAHK may inhibit copper-induced epinephrine degradation by multiple mechanisms. Further studies are warranted to investigate the benecial eect of copper chelation by D DAHK on catecholamine metabolism during sepsis, in an in vivo model.

Acknowledgments
This work was supported by the Trauma Research Department, Swedish Medical Center (Englewood, CO) and DMI BioSciences

A. Roberts et al. / Biochemical and Biophysical Research Communications 304 (2003) 755757 (Englewood, CO). We thank Richard Shimonkevitz, PhD, and Gregory Thomas, BS, for their technical assistance in this research.

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