Juergen Arnhold

Human myeloperoxidase in innate and acquired immunity

Polymorphonuclear leukocytes (PMNs) are important players in innate and acquired immunity. These cells accumulate at inflammatory sites and contribute to host defence, regulation of the inflammatory process, and also to tissue injury. One of the key components of PMNs is the heme-containing enzyme myeloperoxidase (MPO) that is stored in large amount in azurophilic granules of resting cells. Here we review the (patho)physiological role of MPO from the viewpoint of participation of PMNs in immune reactions. Myeloperoxidase is able to catalyse a wide range of one- and two-electron substrate oxidations. With special products, MPO contributes to apoptosis induction in PMNs and other cells, and, thus, to termination of inflammatory response. On the other hand, MPO released from necrotic cells promotes an inflammation by further recruitment of PMNs, and chemical modification of proteins and other tissue constituents. Myeloperoxidase is a fascinating, multifunctional, and challenging enzyme that has not yet revealed all its secrets.

Properties, Functions, and Secretion of Human Myeloperoxidase

The heme-containing protein myeloperoxidase is released from stimulated polymorphonuclear leukocytes at sites of inflammation. It is involved in the generation of reactive oxygen and nitrogen species and tissue damage. The general properties and functional aspects of this enzyme are reviewed. Special attention is given to luminescence methods for investigating the release of myeloperoxidase from stimulated cells.

Experiments towards quantification of saturated and polyunsaturated diacylglycerols by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry

Abstract As recently shown, different physiologically relevant lipid classes can easily be analyzed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI–TOF MS). In the present study the first application of MALDI–TOF for the quantitative analysis of diacylglycerols is described. It is shown that the use of a suitable reference sample enables the quantification of diacylglycerols up to the picomolar range. The best reproducibility of quantitative results for diacylglycerols was obtained using a matrix of 2,5-dihydroxybenzoic acid in ethylacetate and incorporation of an internal standard of the same lipid class. A moderate laser power was used, resulting in a very low extent of fragmentation, allowing a quantification by using solely the highest signal arising from sodium adduct formation of diacylglycerols. A linear correlation between peak intensity and lipid concentration over one order of magnitude was found. The applicability of this new technique for the analysis of other lipids like phosphatidylcholines is also discussed.

Formation of lysophospholipids from unsaturated phosphatidylcholines under the influence of hypochlorous acid

The formation of lysophosphatidylcholines from unsaturated phosphatidylcholines upon treatment with hypochlorous acid was evaluated by means of MALDI-TOF mass spectrometry and 31P NMR spectroscopy. With an increasing number of double bonds in a fatty acid residue, the yield of lysophosphatidylcholines with a saturated fatty acid residue increased considerably in comparison to the total amount of higher molecular weight products like chlorohydrins and glycols. High amounts of lysophosphatidylcholines were formed from phospholipids containing arachidonic or docosahexaenoic acid residues. In phospholipids with monounsaturated fatty acid residues, the position of the double bond did not influence the yield of lyso-products. Besides the exclusive formation of chlorohydrin and glycol, hypochlorous acid caused the cleavage of the unsaturated fatty acid residue independent of its location at the first or second position of the glycerol backbone. In contrast, strong alkaline conditions, i.e. saponification led also to a hydrolysis of the saturated fatty acid residue from phosphatidylcholines. It is concluded that both MALDI-TOF mass spectrometry and 31P NMR spectroscopy are able to detect the formation of lysophosphatidylcholines. We conclude also that the formation of lysophospholipids from unsaturated phosphatidylcholines by hypochlorous acid can be relevant in vivo under acute inflammatory conditions.

THE ACTION OF HYPOCHLOROUS ACID ON PHOSPHATIDYLCHOLINE LIPOSOMES IN DEPENDENCE ON THE CONTENT OF DOUBLE BONDS. STOICHIOMETRY AND NMR ANALYSIS

Kinetics of the consumption of hypochlorous acid in its reaction with double bonds of unsaturated phospholipids and fatty acids were measured using luminol chemiluminescence. Stoichiometry ratios between the consumption of HOCl/OCl- and the loss of double bonds vary from 2:1 to 1:1. Highest values were found in DMPC liposomes containing 5 mol% oleic acid or OPPC. With increasing content of double bonds or higher numbers of double bonds in a fatty acid acyl chain due to incorporated unsaturated fatty acids or phospholipids in DMPC liposomes the stoichiometry ratio falls continuously to 1:1. A ratio of about 1:1 was observed in multilamellar and unilamellar liposomes composed of egg yolk phosphatidylcholine. Products of the reaction of oleic acid with hypochlorous acid were analyses by 1H-NMR spectroscopy. Chlorohydrins were formed in both DMPC liposomes containing 5 or 40 mol% oleic acid.

Deterioration of spermatozoal plasma membrane is associated with an increase of sperm lyso-phosphatidylcholines

Summary.  Spermatozoa with plasma membranes that lost their asymmetry or permeability for larger molecules can be identified by binding of annexin V to membrane phosphatidylserine (PS). Paramagnetic annexin‐V‐conjugated microbeads (AN‐MB) can be used to eliminate these spermatozoa by magnetic activated cell sorting (MACS). Semen samples of six healthy volunteers with normal spermiogram parameters were divided into two sperm fractions by MACS as a function of bound AN‐MB, and their individual lipid compositions were examined by matrix‐assisted laser desorption and ionisation time‐of‐flight mass spectrometry (MALDI‐TOF MS). As a model system, liposomes composed of phosphatidylcholines (PC) from egg yolk were digested by phospholipase A2 (PLA2). The MALDI‐TOF mass spectra of organic extracts of both sperm subpopulations differed significantly. The ratio between lyso‐phosphatidylcholine LPC 16 : 0 and PC 16 : 0/22 : 6 was approximately 2.5–4.7‐fold higher (median 2.9) in the sperm group binding AN‐MB than in spermatozoa with intact membrane unable to bind AN‐MB. The ratio between LPC 22 : 6 and PC 16 : 0/22 : 6 was also enhanced in the spermatozoa with impaired membrane structure (factor in the range: 1.9–3.9; median 2.6). These alterations corresponded to the effects of PLA2 on artificial phospholipids. It is concluded that spermatozoa with deteriorated membrane and exposed PS are characterized by an increased lyso‐phosphatidylcholine content that is likely generated by phospholipases.

Reactivity of cartilage and selected carbohydrates with hydroxyl radicals: an NMR study to detect degradation products.

It was investigated to what extent isolated, monomeric and polymeric carbohydrates as well as cartilage specimens are affected by hydroxyl radicals generated by gamma-irradiation or Fenton reaction and what products can be detected by means of NMR spectroscopy. Resonances of all protons in glucose and other monosaccharides as well as carbon resonances in 13C-enriched glucose were continuously diminished upon gamma-irradiation. Formate and malondialdehyde were found as NMR detectable products in irradiated glucose solutions under physiologically relevant (aerated) conditions. In polysaccharide solutions (e.g. hyaluronic acid) gamma-irradiation and also treatment with the Fenton reagent caused first an enhancement of resonances according to mobile N-acetyl groups at 2.02 ppm. This indicates a breakdown of glycosidic bonds in polysaccharides. Using higher radiation doses or higher concentrations of the Fenton reagent formate was also detected. The same sequence of events was observed upon treatment of bovine nasal cartilage with the Fenton reagent. First, glycosidic linkages in cartilage polysaccharides were cleaved and subsequently formate was formed. In contrast, collagen of cartilage was affected only to a very low extent. Thus, HO-radicals caused the same action on cartilage as on isolated polymer solutions, inducing a fragmentation of polysaccharides and the formation of formate.

Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b−/− mouse model of Wilson's disease

Cellular copper overload as found in Wilsons disease may disturb mitochondrial function and integrity. Atp7b(-/-) mice accumulate copper in the liver and serve as an animal model for this inherited disease. The molecular mechanism of copper toxicity in hepatocytes is poorly understood. Total mitochondrial lipids from liver of wild-type mice were subjected to oxidative stress by the Cu(2+)/H(2)O(2)/ascorbate system. Phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were detected as cardiolipin fragmentation products by thin-layer chromatography combined with MALDI-TOF mass spectrometry in oxidized samples, but not in unperturbed ones. The formation of PA and PHA in copper-treated model membrane correlated well with the decrease of cardiolipin. Mitochondrial lipids from Atp7b(-/-) mice of different age were analyzed for the presence of PA. While 32-weeks old wild-type (control) and Atp7b(-/-) mice did not show any PA, there was a steady increase in the amount of this lipid in Atp7b(-/-) mice in contrast to control with increasing age. Hepatocytes from elder Atp7b(-/-)mice contained morphologically changed mitochondria unlike cells from wild-type animals of the same age. We concluded that free-radical fragmentation of cardiolipin with the formation of PA is a likely mechanism that damages mitochondria under conditions of oxidative stress due to copper overload. Our findings are relevant for better understanding of molecular mechanisms for liver damage found in Wilsons disease.

Modulation of luminol chemiluminescence of fMet-Leu-Phe-stimulated neutrophils by affecting dephosphorylation and the metabolism of phosphatidic acid.

This paper is addressed to study how PKC-mediated effects and phosphatidic acid interact together in activation of NADPH-oxidase in formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) stimulated neutrophils as detected by luminol chemiluminescence. The early luminescence response in fMet-Leu-Phe-stimulated cells (up to 5 min after stimulation) depends mainly on reactive oxygen species generated extracellularly, whereas all later events are caused by oxidation of luminol inside the cells. The two protein phosphatase inhibitors, okadaic acid and calyculin A, dramatically increased the late luminescence of cells. This enhancement was totally inhibited by the phospholipase D modulator butanol, while the protein kinase C (PKC) inhibitor bisindolylmaleimide I was insensitive. The early luminescence response of the cells was slightly inhibited by both protein phosphatase inhibitors and depended on protein kinase C as well as on phospholipase D activities. Propranolol, an inhibitor of phosphatidate phosphohydrolase, enhanced all parts of luminescence response of fMet-Leu-Phe-stimulated neutrophils at concentrations up to 2.5 x 10(-5) mol/L. While the late luminescence response of propranolol-treated cells was not inhibited by the PKC inhibitor bisindolylmaleimide I, the first response depended on protein kinase C. The inhibitor of diacylglycerol kinase R59949 enhanced the luminescence signal only during the first 4 min in fMet-Leu-Phe-stimulated cells. Only diacylglycerols derived from phospholipase C, such as 1-stearoyl-2-arachidonoyl-sn-glycerol, were able to initiate an oxidative burst in cells. Saturated diacylglycerols (e.g. 1,2-dipalmitoyl-sn-glycerol or 1,2-distearoyl-sn-glycerol) did not yield any luminol chemiluminescence, although they were incorporated into the plasma membrane, as evidenced by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Our results demonstrate that phosphatidic acid produced by phospholipase D is responsible for NADPH-oxidase activity in fMet-Leu-Phe-stimulated neutrophils over the entire measuring time, whereas PKC-mediated processes are only involved during the first 5 min.

Differences in the reactivity of phthalic hydrazide and luminol with hydroxyl radicals

The reactivity of 5-amino-2,3-dihydro-phthalazine-1,4-dione (luminol) and phthalic hydrazide with hydroxyl radicals was studied. HO*-radicals were generated by the Fenton reaction as well as by water radiolysis. Both luminol and phthalic hydrazide react with hydroxyl radicals under intense chemiluminescence (CL) emission. However, exclusively the CL arising from phthalic hydrazide oxidation can be quenched by competition (e.g. by the addition of carbohydrates), whereas luminol CL is enhanced. The reactivities of both compounds with HO*-radicals were further studied by time-resolved spectroscopy (pulse radiolysis), competition methods, NMR spectroscopy and mass spectrometry. Whereas only slight differences were detectable by pulse radiolysis, the analysis of competition kinetics in the presence of p-nitroso-dimethylaniline (NDMA) gave a two-fold-enhanced reactivity for luminol (4.8 x 10(9) l mol(-1) s(-1)) in comparison to phthalic hydrazide (2.0 x 10(9) l mol(-1) s(-1)). NMR and mass spectrometric analyses revealed significant differences in the reactivity of HO*-radicals: whereas in luminol solutions hydroxylation of the aromatic ring system predominated, hydroxylated products were not detectable upon irradiation of phthalic hydrazide. A hypothetical mechanism is proposed which may explain the observed differences.