Selective pick-up of increased iron by deferoxamine-coupled cellulose abrogates the iron-driven induction of matrix-degrading metalloproteinase 1 and lipid peroxidation in human dermal fibroblasts in vitro: a new dressing concept.

Using atomic absorption spectrum analysis, we found iron levels in exudates from chronic wounds to be significantly increased (3.71 +/- 1.56 micromol per g protein) compared to wound fluids from acute wounds derived from blister fluids (1.15 +/- 0.62 micromol per g protein, p < 0.02), drainage fluids of acute wounds (0.87 +/- 0.34 micromol per g protein, p < 0.002), and pooled human plasma of 50 volunteers (0.42 micromol per g protein). Increased free iron and an increase in reactive oxygen species released from neutrophils represent pathogenic key steps that --via the Fenton reaction - are thought to be responsible for the persistent inflammation, increased connective tissue degradation, and lipid peroxidation contributing to the prooxidant hostile microenvironment of chronic venous leg ulcers. We herein designed a selective pick-up dressing for iron ions by covalently binding deferoxamine to cellulose. No leakage occurred following gamma sterilization of the dressing and, more importantly, the deferoxamine-coupled cellulose dressing retained its iron complexing properties sufficient to reduce iron levels found in chronic venous ulcers to levels comparable to those found in acute wounds. In order to study the functionality of the dressing, human dermal fibroblasts were exposed to a Fenton reaction mimicking combination of 220 microM Fe(III) citrate and 1 mM ascorbate resulting in a 4-fold induction of matrix-degrading metalloproteinase 1 as determined by a matrix-degrading metalloproteinase 1 specific enzyme-linked immunosorbent assay. This induction was completely suppressed by dissolved deferoxamine at a concentration of 220 microM or by an equimolar amount of deferoxamine immobilized to cellulose. In addition, the Fe(III) citrate and ascorbate driven Fenton reaction resulted in an 8-fold increase in malondialdehyde, the major product of lipid peroxidation, as determined by high pressure liquid chromatography. This increase in malondialdehyde levels could be significantly reduced in the presence of the selective pick-up dressing coupled with deferoxamine suggesting that the deferoxamine dressing, in fact, prevents the development of a damaging prooxidant microenvironment and also protects from unfavorable consequences like matrix-degrading metalloproteinase 1 and lipid peroxide induction.

Hydroactive dressings and serum proteins: an in vitro study.

An in vitro approach was used to obtain information about the compatibility of hydroactive dressing materials with the serum proteins which are thought to be relevant to wound healing. Artificial wound fluid was incubated with different hydroactive dressings (Cutinova hydro, Varihesive E, Comfeel Ulcer Dressing and Allevyn), and concentrations of total protein, albumin, immunoglobulin and growth factors were measured after one day of incubation. Cutinova hydro and Allevyn absorbed considerable amounts of fluid. Fluid uptake was lower for the hydrocolloid dressings. An unexpected finding was that Cutinova hydro showed an approximately two-fold increase over control values in the concentration of all proteins tested, indicating a selective absorption of water by this dressing. For the other dressings tested, zero or very low absorption of proteins was found, indicating a basically satisfactory protein compatibility.

Hydroactive dressings and serum proteins: an in vitro study.

An in vitro approach was used to obtain information about the compatibility of hydroactive dressing materials with the serum proteins which are thought to be relevant to wound healing. Artificial wound fluid was incubated with different hydroactive dressings (Cutinova hydro, Varihesive E, Comfeel Ulcer Dressing and Allevyn), and concentrations of total protein, albumin, immunoglobulin and growth factors were measured after one day of incubation. Cutinova hydro and Allevyn absorbed considerable amounts of fluid. Fluid uptake was lower for the hydrocolloid dressings. An unexpected finding was that Cutinova hydro showed an approximately two-fold increase over control values in the concentration of all proteins tested, indicating a selective absorption of water by this dressing. For the other dressings tested, zero or very low absorption of proteins was found, indicating a basically satisfactory protein compatibility.

Metabolism of ether lysophospholipids in Leishmania donovani promastigotes.

Ether lysophospholipids, 1-O-[1'-14C]octadec-1'-enyl-sn-glycero-3-phosphoethanolamine (A) and -phosphocholine (B) as well as 1-O-[1'-14C]octadecyl-sn-glycero-3-phosphoethanolamine (C) and -phosphocholine (D) were taken up rapidly and metabolized extensively in Leishmania donovani promastigotes. Degradation to neutral lipids occurred first, followed by incorporation into phospholipids. Incubation of the cells with (A) or (B) revealed the stability of the O-[1-14C]octadec-1-enyl group up to 15 h, indicating the absence of any O-alk-1-enyl cleavage enzymes. Most of the radioactivity was found in 1-O-alkenyl-2-acyl-glycerophosphoethanolamine and 1-O-alkenyl-2,3-diacylglycerol. 1-O-Alkenyl-2-acyl-glycerophosphocholine was detected only after incubation with substrate (B). In contrast to the O-alk-1-enyl residue, the O-[1-14C]octadecyl moiety in substrate (C) and (D) could be converted into the O-[1-14C]octadec-1-enyl moiety or cleaved, yielding labelled acyl groups. Following 5 h incubation with substrate (C), most of the incorporated radioactivity was associated with 1-O-[1'-14C]octadec-1'-enyl-2-acyl-glycerophosphoethanolamine, 1-O-[1'-14C]octadecyl-2,3-diacylglycerol and 1-O-[1'-14C]octadecyl-2-acyl-glycerophosphoinositol. After 15 h minor amounts of label appeared in diacyl glycerophosphocholine. Similar labelling patterns were obtained with the choline analogue (D), except that 1-O-[1'-14C]octadecyl-2-acyl-glycerophosphocholine was found additionally. Incubations of the four labelled ether lysophospholipids with cell homogenates showed the presence of a lysophospholipase D and a phosphohydrolase. There was no specificity towards different ether residues or phosphobase moieties. Formation of alkyl- and alkenylglycerol, respectively, was stimulated by Mg2+ ions and the phosphohydrolase was inhibited by NaF. The results support the conclusion that the specific pattern of ether phospholipids in L. donovani cells is due to a pronounced specificity of the biosynthetic enzymes. Enzymes of the catabolic reactions are of low specificity or absent, such as plasmalogenases.

Cytotoxicity of ester and ether lysophospholipids on Leishmania donovani promastigotes.

The cytotoxic activity of four ester lysophospholipids, three ether lysophospholipids, and two radylglycerols on Leishmania donovani promastigotes was determined by measuring the inhibition of cell growth. The 1-acyl lysophospholipids reduced cell growth to 50% of controls at concentrations of 6.4-10.9 microM. In contrast, 1-O-alkenyl-sn-glycero-3-phosphoethanolamine, 1-O-hexadecyl-sn-glycero-3-phosphocholine, and 1-O-hexadecyl-sn-glycerol already showed a 50% inhibition of growth at concentrations between 2.1 and 2.8 microM. Moreover, the unnatural alkyl lysophospholipid analogue 1-O-octadecyl-2-methoxy-sn-glycero-3-phosphocholine was even 10-fold more toxic. Incubations of L. donovani promastigotes with radioactively labelled ether lysophospholipids revealed a rapid uptake of these compounds and their incorporation into cellular lipids at a non-toxic concentration of 1.0 microM. An accumulation of the lysophospholipids in the cell due to insufficient metabolism may be the cause of its cytotoxic effect. The sensitivity of L. donovani cells towards ether lysophospholipids was found to be similar to that reported for tumor cells.

Conversion of radiolabelled ethanolamine plasmalogen into the dimethylethanolamine and choline analogue via transphosphatidylation by phospholipase D from cabbage.

Starting from biosynthetically prepared ethanolamine plasmalogen 14C-labelled in the O-alkenyl moiety, choline and dimethylethanolamine plasmalogen were prepared by transphosphatidylation utilizing phospholipase D from cabbage. Investigation of the time course of the reaction showed that transphosphatidylation was simultaneously accompanied by hydrolysis of both the substrate and the desired product, resulting in a maximum of product yield after 1-3 h under the reaction conditions investigated. Optimal reaction conditions gave yields of 40% and 62% (of total radioactivity) respectively for the purified choline and dimethylethanolamine derivatives.

Biosynthetic preparation of radioactively labelled ethanolamine plasmalogen (1-O-[1'-14C]octadec-1'-enyl-2-acyl-sn-glycero-3-phosphoethanola mine) using a protozoan cell culture.

Ethanolamine plasmalogen radiolabelled mainly in the O-alkenyl moiety was prepared from cell suspension cultures of the flagellate Leishmania donovani previously incubated with [1-14C]octadecanol over one growth period. The optimal concentration of [1-14C]octadecanol for labelling was shown to be 1 microM, when 60% of total lipid radioactivity appeared in the 1,2-diradyl-sn-glycero-3-phosphoethanolamine fraction, with an overall yield of approx. 35%. Analysis of this fraction revealed that 93% of the label was present in O-octa-dec-1-enyl, 3% in O-alkyl and 4% in acyl moieties. A specific radioactivity of approx. 14 mCi/mmol was determined. Raising the culture medium concentration of [1-14C]octadecanol to 2 microM yielded a product with a specific radioactivity of 25 mCi/mmol.