Holger Spalteholz

Effects of hypochlorous acid on unsaturated phosphatidylcholines.

Effects of hypochlorous acid and of the myeloperoxidase-hydrogen peroxide-chloride system on mono- and polyunsaturated phosphatidylcholines were analyzed by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Chlorohydrins and glycols were detected as main products according to the characteristic shift of molecular masses. Mainly mono-chlorohydrins result upon the incubation of HOCl/(-)OCl with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, whereas only traces of mono-glycols were detected. 1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine yielded a complex mixture of products. Mono-chlorohydrins and glycols dominated only at short incubation, while bis-chlorohydrins as well as products containing one chlorohydrin and one glycol moiety appeared after longer incubation. Similarly, a complex product mixture resulted upon incubation of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine with hypochlorous acid. Additionally, tris-chlorohydrins, products with two chlorohydrin and one glycol moiety, as well as lysophosphatidylcholines and fragmentation products of the arachidonoyl side chain were detectable. Mono-chlorohydrins of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine were detected after the incubation of the latter phospholipid with the myeloperoxidase-hydrogen peroxide-chloride system at pH 6.0. These chlorohydrins were not observed in the absence of chloride, hydrogen peroxide, or myeloperoxidase as well as in the presence of methionine, taurine, or sodium azide. Thus, mono-chlorohydrins in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine produced by hypochlorous acid from the myeloperoxidase-hydrogen peroxide-chloride system can also be detected by means of MALDI-TOF MS.

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.

Myeloperoxidase-induced formation of chlorohydrins and lysophospholipids from unsaturated phosphatidylcholines

The formation of lysophosphatidylcholines and chlorohydrins from unsaturated phosphatidylcholines upon the treatment with the myeloperoxidase-hydrogen peroxide-chloride system was evaluated by means of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Lyso-products were primarily found in phosphatidylcholine samples containing highly unsaturated fatty acid residues such as arachidonic or docosahexenoic acid. On the other hand, chlorohydrins dominate in mono- or bis-unsaturated phosphatidylcholines. No formation of these products was detected in the absence of one of the components of the MPO-H(2)O(2)-Cl(-) system or in the presence of MPO inhibitors (sodium azide) or scavengers of hypochlorous acid (taurine, methionine). Thus, hypochlorous acid formed by the MPO-H(2)O(2)-Cl(-) system is responsible for the observed modification in unsaturated phosphatidylcholines. In the presence of the complete MPO system, lyso-products and chlorohydrins were only formed at pH values lower than pH 6.0 with an optimum at pH 4.3. In contrast, the reagent hypochlorous acid caused the formation of these products even at neutral pH values, indicating a clear dependence of the yield of products on the presence of undissociated HOCl. We conclude that the formation of lysophospholipids and chlorohydrins from unsaturated phosphatidylcholines by myeloperoxidase can be relevant in vivo under acute inflammatory conditions.

Myeloperoxidase binds to non-vital spermatozoa on phosphatidylserine epitopes

The heme protein myeloperoxidase is released from stimulated polymorphonuclear leukocytes, a cell species found in increasing amounts in the male and female genital tract of patients with genital tract inflammations. Myeloperoxidase binds only to a fraction of freshly prepared human spermatozoa. The number of spermatozoa able to bind myeloperoxidase raised considerably in samples containing pre-damaged cells or in acrosome-reacted samples. In addition, myeloperoxidase released from zymosan-stimulated polymorphonuclear leukocytes was also able to bind to pre-damaged spermatozoa. The ability of spermatozoa to bind myeloperoxidase coincided with the binding of annexin V to externalized phosphatidylserine epitopes indicating the loss of plasma membrane integrity and with the incorporation of ethidium homodimer I. Myeloperoxidase did not interact with intact spermatozoa. Annexin V and myeloperoxidase bind to the same binding sites as verified by double fluorescence techniques, flowcytometry analyses as well as competition experiments. We demonstrated also that myeloperoxidase is eluted together with pure phosphatidylserine liposomes or liposomes composed of phosphatidylserine and phosphatidylcholine in gel filtration, but not with pure phosphatidylcholine liposomes. In conclusion, myeloperoxidase interacts with apoptotic spermatozoa via binding to externalized phosphatidylserine indicating a yet unknown role of this protein in recognition and removal of apoptotic cells during inflammation.

Modification of phosphatidylserine by hypochlorous acid

The binding of the heme enzyme myeloperoxidase to phosphatidylserine epitopes on the surface of non-vital polymorphonuclear leukocytes and other cells at inflammatory sites favours modifications of this phospholipid by myeloperoxidase products. As detected by MALDI-TOF mass spectrometry hypochlorous acid and the myeloperoxidase-hydrogen peroxide-chloride system convert 1,2-dipalmitoyl-sn-glycero-3-phosphoserine into 1,2-dipalmitoyl-sn-glycero-3-phosphoacetaldehyde and 1,2-dipalmitoyl-sn-glycero-3-phosphonitrile. A transient chlorimine derivative was detected using 4-chloro-alpha-cyanocinnamic acid as matrix in mass spectrometry only at short incubation times and supplying HOCl in two-fold excess. The decay of transient chlorinated products was followed by changes in absorbance spectra using O-phospho-l-serine to model the behavior of the serine head group in phosphatidylserine. N-Chlorimine and N-monochloramine derivatives decayed with half-life times of 1.5 and 57 min, respectively, at 22 degrees C and pH 7.4. N-Dichloramines decayed within few seconds under these conditions.

Leukocytic myeloperoxidase-mediated formation of bromohydrins and lysophospholipids from unsaturated phosphatidylcholines

Using MALDI-TOF mass spectrometry, we have shown that leukocytic myeloperoxidase (MPO) in the presence of its substrates (H2O2 and Br−) does not induce any changes in saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Incubation of liposomes prepared from mono-unsaturated phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) with the (MPO + H2O2 + Br−) system resulted in formation of bromohydrins as the main products. 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysophosphatidylcholine) was the main product of the reaction of polyunsaturated phosphatidylcholine (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) with the (MPO + H2O2 + Br−) system. The formation of lysophospholipids as well as of bromohydrins was not observed when the enzyme or one of its substrates (H2O2 or Br−) was absent from the incubation medium, or if an inhibitor of MPO (sodium azide) or hypobromite scavengers (taurine or methionine) were added. Thus, it can be postulated that the formation of bromohydrins as well as lysophospholipids by the (MPO + H2O2 + Br−) system results from reactions of hypobromite formed during MPO catalysis with double bonds of acyl chains of phosphatidylcholine. Such destructive processes may take place in vivo in membrane-or lipoprotein-associated unsaturated lipids in centers of inflammation.