Metabolism of 4-hydroxy-2-nonenal in human polymorphonuclear leukocytes

doi:10.1016/j.abb.2010.08.018

Archives of Biochemistry and Biophysics

Volume 503, Issue 2, 15 November 2010, Pages 248-252

https://doi.org/10.1016/j.abb.2010.08.018 Get rights and content

Abstract

Intracellular metabolism of 4-hydroxy-2-nonenal (HNE), a major product and mediator of oxidative stress and inflammation, is analyzed in resting and fMLP-stimulated human polymorphonuclear leukocytes (PMNL), where this compound is generated during activation of the respiratory burst. HNE consumption rate in PMNL is very low, if compared to other cell types (rat hepatocytes, rabbit fibroblasts), where HNE metabolism is always an important part of secondary antioxidative defense mechanisms. More than 98% of HNE metabolites are identified. The pattern of HNE intermediates is quite similar in stimulated and resting PMNL – except for higher water formation in resting PMNL – while the initial velocity of HNE degradation is somewhat higher in resting cells, 0.44 instead of 0.28 nmol/(min × 10⁶ cells). The main products of HNE metabolism are 4-hydroxynonenoic acid (HNA), 1,4-dihydroxynonene (DHN) and the glutathione adducts with HNE, HNA, and DHN. Protein-bound HNE and water account for about 3–4% of the total HNE derivatives in stimulated cells, while in resting cells protein-bound HNE and water are 4% and 20%, respectively. Cysteinyl-glycine-HNE adduct and mercapturic acids contribute to about 5%.

Research highlights

► HNE degradation rate by human PMNL is very low, if compared with other cells. ► HNE degradation rate is even lower in the activated cells. ► GSH-HNE adduct and related carboxylic acids and alcohols are the main metabolites. ► Water and carbon dioxide were identified as secondary products.

Introduction

Lipid peroxidation is combined with the formation of reactive aldehydes [1], [2]. 4-Hydroxy-2-nonenal (HNE)¹ is a major aldehyde produced in vivo during peroxidation of omega-6-polyunsaturated fatty acids (18:2, 20:4) [1], [3], [4]. A primary involvement of this aldehydic product of membrane lipid peroxidation in inflammation-related events, as well as in regulation of cell proliferation and growth and in apoptotic cell death, appears to be supported by its marked ability to modulate several major pathways of cell signaling and gene expression [4], [5], [6].

HNE is both a product and a regulating factor of phagocytosis by polymorphonuclear leukocytes (PMNL). Years ago, HNE has been found to stimulate the oriented migration of PMNL at concentrations ranging from 10⁻⁷ to 10⁻⁵ M [7], [8], [9]. The chemotactic activity of HNE might be mediated by the stimulation of phosphoinositide-specific phospholipase C [10]. Only at concentrations higher than 500 μM HNE inhibited PMNL motility, whereas significant inhibition of phospholipase C activity occurred at 100 μM [10]. The NADPH oxidase activity is affected already at HNE levels of about 10 μM with a 50% inhibition at about 20 μM HNE [11], [12]. If the level of HNE is increased in organs or tissues, an increased number of PMNL is moving to these areas [9].

It is generally accepted that HNE levels are increased in inflammatory regions [4]. It was shown that HNE and resulting HNE-protein adducts are generated as a result of NADPH oxidase activity in the phagosomes of human neutrophils. It was concluded that these lipid peroxidation (LPO) products contribute to microbial killing and/or damage of neutrophil phagolysosomal proteins [13].

The metabolism of HNE is a secondary antioxidative mechanism diminishing the binding of HNE to cysteine, lysine and histidine residues of proteins [14]. HNE metabolism was analyzed in various cells, tissues and organs [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. The HNE metabolism seems to represent very rapid pathways that are able to diminish even high HNE concentrations, which are exogenously added to cells or tissues, within a few minutes [19]. End products of HNE metabolism were proposed to be biomarkers of in vivo lipid peroxidation [4], [25], [26]. Influence on HNE metabolism may be of clinical therapeutic interest [27], [28], [29], [30].

In this study the capacity of HNE degradation and the different pathways of HNE metabolism in human PMNL were measured. The studies were carried out with human PMNL during their resting phase and with cells that were stimulated by fMLP. The velocity of HNE metabolism was unusually low in PMNL compared with all other cell types, tissues, and organs, which have been investigated up to now.

Section snippets

Materials

HNE was supplied as HNE diethyl acetal dissolved in chloroform and was stored at −20 °C until required. Just prior to use, HNE was prepared from its diethyl acetal derivative by 1 mM HCl hydrolysis at room temperature [31], [32]. [2-³H]-HNE was prepared according to Rees et al. [33]. The radiochemical purity of [2-³H]-HNE, determined by HPLC, was 96.5%. The specific radioactivity was 68.2 mCi/mmol.

The standards for TLC were prepared as described [16]. All other chemicals were purchased from Merck

Results

Fig. 1 shows the time course of degradation of 10 μM [2-³H]-HNE by resting and fMLP-stimulated human PMNL. After 1 min incubation, 22% or 14% of labeled HNE (0.44 or 0.28 nmol/10⁶ cells) were degraded by resting or stimulated cells, respectively. Such a rate of HNE degradation amounts to only one hundredth of that in other cell types of similar volume, like hepatocytes or fibroblasts, indicating that the rate of HNE metabolism in PMNL is unexpectedly low (Table 1). After 30 min incubation, 13% or

Discussion

Schematic representation of HNE metabolic pathways, which have been described in literature up to now [14], [19], [21], [37], and HNE degradation products, which are identified in this study in human PMNL, are reported in Fig. 4.

The HNE adduct with glutathione (GSH), the corresponding carboxylic acid of HNE (HNA), and the corresponding alcohol (DHN) are the main primary metabolites of HNE. Neutrophils have a significant content of GSH and a significant amount of transferase activity [38], which

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Metabolism of 4-hydroxy-2-nonenal in human polymorphonuclear leukocytes

Abstract

Research highlights

Introduction

Section snippets

Materials

Results

Discussion

Free Radic. Biol. Med.

Biochim. Biophys. Acta

Mol. Aspects Med.

Mol. Aspects Med.

J. Lipid Res.

Free Radic. Biol. Med.

Mol. Aspects Med.

Free Radic. Biol. Med.

Chem. Biol. Interact.

J. Biol. Chem.

J. Immunol. Methods

Free Radic. Biol. Med.

J. Lipid Res.

J. Biol. Chem.

Arch. Biochem. Biophys.

Chem. Biol. Interact.

Biochem. Pharmacol.

Mol. Aspects Med.

Mutat. Res.

IUBMB Life

Med. Res. Rev.

Acta Biochim. Pol.

Res. Commun. Chem. Pathol. Pharmacol.

Biomed. Pharmacother.

Free Radical Res.