HPLC-fluorimetric assay of phospholipase A2. Application to biological samples with high protein content and various reaction conditions.

Phospholipase A(2) (PLA(2)) quantitation in real-time, using (7-nitro-2-1,3-benzoxadiazol-4-yl)amino-derivatives of phosphatidylcholine (NBD-PCs) as substrates, is influenced by high protein content, color or turbidity. The purpose of the study was to overcome such limitations by a complementary reversed-phase HPLC step to separate the substrates from the products of the reaction. Plasma and bronchoalveolar lavage (BAL) fluid were applied as enzymic sources, while standard porcine PLA(2) and human plasma PAF-acetylhydrolase (PAF-AH) were employed as positive controls. The method was validated with a radiometric assay and compared with the real-time fluorimetric assay. Regarding PLA(2) and PAF-AH determination, the isocratic elution systems CH(3)OH-H(2)O (80:20, v/v) and CH(3)OH-H(2)O-CH(3)COOH (60:40:0.2, v/v/v) separated efficiently C(12)-NBD-FA/C(12)-NBD-PC and C(6)-NBD-FA/C(6)-NBD-PC, with 4.4 and 2.2 resolution, respectively. Analysis time was ∼15 min/injection. The reproducibility, expressed as relative standard deviation, was ≤13% for PLA(2) and ≤16% for PAF-AH, respectively. The assay was linear for PLA(2) activities extending from 1 pmol up to at least 250 nmol FA/h/mL sample, similar to the radiometric assay. It was appropriate for samples with high protein content, where the real-time fluorimetric assay was insufficient. The HPLC method was also evaluated under elevated temperatures, various pH values and Ca(2+) concentrations.

Desferrioxamine attenuates minor lung injury following surgical acute liver failure.

Acute liver failure (ALF) can be complicated by lung dysfunction. The aim of this study was to test the hypothesis that inhibition of oxidative stress through iron chelation with desferrioxamine (DFX) attenuates pulmonary injury caused by ALF. 14 adult female domestic pigs were subjected to surgical devascularisation of the liver and were randomised to a study group (DFX group, n = 7), which received post-operative intravenous infusion of DFX (14.5 mg x kg(-1) x h(-1) for the first 6 h post-operatively and 2.4 mg x kg(-1) x h(-1) until completion of 24 h), and a control group (n = 7). Post-operative lung damage was evaluated by histological and bronchoalveolar lavage fluid (BALF) analysis. DFX resulted in reduced BALF protein levels and tissue phospholipase (PL)A(2) activity. Plasma malondialdehyde and BALF nitrate and nitrite concentrations were lower, while catalase activity in the lung was higher after DFX treatment. PLA(2), platelet-activating factor acetylhydrolase and total cell counts in BALF did not differ between groups. Histological examination revealed reduced alveolar collapse, pneumonocyte necrosis and total lung injury in the DFX-treated animals. DFX reduced systemic and pulmonary oxidative stress during ALF. The limited activity of PLA(2) and the attenuation of pneumonocyte necrosis could represent beneficial mechanisms by which DFX improves alveolar-capillary membrane permeability and prevents alveolar space collapse.

Acute lung injury probably associated with infusion of propofol emulsion.

We present a case of acute lung injury associated with propofol infusion in a mechanically ventilated patient with intracerebral haemorrhage. Diagnosis was based on the exclusion of other risk factors inducing acute lung injury and on the clinical improvement after discontinuation of the propofol emulsion. Laboratory data such as the increase in total phospholipids, neutral lipids and free fatty acids in the broncho-alveolar lavage fluid, the remarkably high percentage of alveolar macrophages including fat droplets and the similar lipid composition of propofol and broncho-alveolar lavage fluid support the relationship between propofol and acute lung injury.

Anti-phospholipid antibodies in serum from patients with Guillain-Barré syndrome.

OBJECTIVE: The Guillain-Barré syndrome (GBS) is an acute inflammatory polyneuropathy related to autoimmunity. However, no conclusive etiological concept has yet been found. We examined the variation in autoantibodies to lipids in serum of GBS patients in response to the course of the disease but investigated titer modifications during treatment with gamma-globulin. DESIGN AND SETTING: Prospective clinical study in a 14-bed general ICU. PATIENTS: Nine patients with GBS and nine controls were included in the study. MEASUREMENTS AND RESULTS: Four blood samples were obtained before and after treatment. Serum samples, diluted 1:60, were tested by enzyme-linked immunosorbent assay for IgM, IgA, and IgG antibodies to phosphatidylcholine, phosphatidylinositol, cardiolipin, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, and gangliosides. Anti-phospholipid antibodies of the IgM, IgA, and IgG families were detected in all GBS patients but in none of the controls. Phosphatidylinositol, cardiolipin, phosphatidylcholine, and phosphatidic acid were the main antigens. All patients developed anti-phosphatidylinositol antibodies of the IgM family and anti-cardiolipin antibodies of the IgA and IgG families. A decrease in the level of anti-phospholipid autoantibodies was observed after 1 day of treatment with gamma-globulin. Two days after ending gamma-globulin administration the IgG antibodies increased again. CONCLUSIONS: Our findings suggest that in GBS there is an extensive immune reaction, which is altered after gamma-globulin treatment. Anti-cardiolipin and anti-phosphatidylinositol antibodies could be useful markers for the response to treatment.

Bronchoalveolar lavage alterations during prolonged ventilation of patients without acute lung injury.

Mechanical ventilation deteriorates previously injured lung, but little is known about its effect on healthy human lung. This work was designed to assess the effect of prolonged mechanical ventilation on bronchoalveolar lavage (BAL) fluid composition of patients without acute lung injury. Twenty-two ventilated patients (tidal volume 8-10 mL x kg(-1), positive end-expiratory pressure 3-5 cmH2O) without lung injury, who did not develop any complication from the respiratory system during the 2-week study period, were studied. They were subjected to three consecutive BALs, the first during 36 h from intubation, the second at the end of the first week of mechanical ventilation and the third at the end of the second week of mechanical ventilation. Total BAL protein increased during mechanical ventilation (148 +/- 62, 381 +/- 288, 353 +/- 215 microg x mL(-1) BAL for the first, second and third BAL, respectively). In contrast, BAL phospholipids decreased (2.7 +/- 1.1, 1.4 +/- 0.6, 1.2 +/- 0.7 microg x mL(-1) BAL, respectively). Large surfactant aggregates were reduced and inflammatory markers, such as platelet activating factor (PAF), PAF-acetylhydrolase and neutrophils, significantly increased after 1 week, but partially remitted after 2 weeks of mechanical ventilation. In summary, this study demonstrates that prolonged mechanical ventilation even of patients without acute lung injury is associated with the presence of inflammatory markers and surfactant alterations.

Ventilator-associated pneumonia and atelectasis: evaluation through bronchoalveolar lavage fluid analysis.

OBJECTIVE: Surfactant offers protection against alveolar collapse and contributes to the local defense mechanism, but it is unclear if surfactant alterations have a role in the development of atelectasis or ventilator-associated pneumonia (VAP). The present study was undertaken to monitor surfactant, as well as biochemical BAL fluid alterations, during the course of VAP and atelectasis in mechanically ventilated patients without primary cardiopulmonary disease, to elucidate the pathogenesis and to differentiate these two entities. DESIGN. Prospective controlled study. SETTING: 14-bed general ICU of a 750-bed University Hospital. PATIENTS: Sixty-one ventilated patients, without primary cardiopulmonary disease-normal initial chest X-ray, satisfactory oxygenation (PaO(2)/FiO(2)>300 mmHg), and expected time of ventilation exceeding 2 weeks-were initially enrolled. Twelve of them developed VAP and eight lobar or segmental atelectasis during the 2-week study period. INTERVENTIONS: An initial BAL was performed in all patients within 48 h from admission. Patients who developed VAP or atelectasis were subjected to a second and third BAL during and after the resolution of VAP or atelectasis, respectively. MEASUREMENTS AND RESULTS: VAP and atelectasis resulted in a significant increase of total protein and markers of inflammation, such as PAF and neutrophils, which partially remitted after their resolution. Large surfactant aggregates, which contribute to surface tension decrease, were significantly reduced during both entities and remained low even after their resolution. CONCLUSIONS: BAL alterations during VAP and atelectasis suggest increased alveolar-capillary permeability, severe surfactant abnormalities, and signs of local inflammatory reaction. These alterations are associated with the observed deteriorated gas exchange and lung mechanics and could predispose to further lung injury in ventilated patients.

Studies on the Paramecium aurelia species complex in Lake Pamvotis (Ioannina, N.W. Greece).

Paramecium sexaurelia was identified in Lake Pamvotis, Grecce. The occurrence of this species there was confirmed.

1-O-alkyl-2-acetyl-sn-glycero-3-phosphate:phosphohydrolase activity in Tetrahymena pyriformis.

Within the frame of the de novo formation of Platelet-Activating Factor in Tetrahymena, the occurrence as well as the properties of a lipid phosphate phosphohydrolase activity catalyzing the dephosphorylation of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate was investigated. The activity was distributed in all the membrane fractions of the cell and in the cytosol. It showed preference for acyl-acetyl-sn-glycero-phosphate as well, and at a much lower level, for dipalmitoyl-glycero-phosphate. Mg2+ and Ca2+ caused a dose-dependent inhibition, while F-, EDTA and EGTA had no effect. The enzymic activity was linear for at least up to 60 min incubation time and up to 150 microg protein. Microsomal activity exhibited two optimal pH areas, around 7.0 and 9.0, while mitochondrial activity showed one peak, at pH 7.0. Acyl-GP, acyl-acetyl-GP and alkyl-GP could replace alkyl-acetyl-GP in significant rates, while dipalmitoyl-GP, beta-GP, fructose-6-GP, p-nitrophenylphosphate, creatine phosphate or ATP had no effect. Side phospholipase A2 and C activities were also detected. Taking into account the presence of PAF and alkylacetylglycerol in the protozoan as well as the presence of a dithiothrcitol-insensitive CDP-choline:cholinephosphotransferase activity that converts alkylacetylglycerol to PAF, we suggest that the present phosphohydrolase activity may be involved in the de novo production of PAF within Tetrahymena.

Differential determination of phospholipase A(2) and PAF-acetylhydrolase in biological fluids using fluorescent substrates.

The purpose of the present study was the development and evaluation of a fluorimetric method for the screening and differential determination of phospholipase A(2) and PAF-acetylhydrolase in bronchoalveolar lavage (BAL) fluid and serum. Phospholipase A(2) was determined using C(12)-NBD-PC in the presence of Ca(2+), from the slope of the fluorescence enhancement due to the formation of C(12)-NBD-fatty acid. PAF-acetylhydrolase was determined using C(6)-NBD-PC, from the slope of the curve due to C(6)-NBD-fatty acid formation in the absence of Ca(2+). The results were confirmed after TLC analysis. The method's selectivity was evaluated by comparing to radiometric measurements. Light scattering did not interfere and inner filter effects was not observed under our experimental conditions. The effects of pH, temperature, and Ca(2+) were investigated. Protein caused an increase in the background fluorescence of both NBD-PCs. The standard curves of both NBD-fatty acids exhibited the same slope. Linearity extended at least up to 4. 5 nmoles per ml of reaction mixture at the normal pH 7.4. The fluorescence of the NBD-fatty acids remained stable for increasing concentrations of BAL fluid and serum and for BSA up to 100 microg/ml of reaction mixture. Porcine pancreatic PLase A(2) showed preference for C(12)-NBD-PC in the presence of Ca(2+), while without Ca(2+), serum PAF-AcH hydrolyzed only C(6)-NBD-PC. The method is highly sensitive, accurate, and reproducible and can be applied for the differential determination of phospholipase A(2) and PAF-acetylhydrolase activities in BAL fluid and serum.

Bronchoalveolar lavage alterations in pulmonary embolism.

The objective of this study was to determine quantitative and qualitative surfactant alterations, proteins, and platelet activating factor (PAF) in bronchoalveolar lavage (BAL) fluid from patients with pulmonary thromboembolism (PTE) with respect to ventilated patients without PTE. Patients with PTE underwent BAL at the most affected lung area on the first and tenth days of PTE diagnosis. Total proteins and albumin, total lipids, individual phospholipid classes, PAF and PAF-acetylhydrolase (PAF-AcH) activity were determined in BAL fluid. Total proteins and albumin were found to be increased in both successive samples of patients with PTE when compared with the control group (p < 0.001 and p < 0.05, respectively). Total phospholipids, though, were elevated on the first day, but they decreased on the tenth day, in comparison with the control groups (p < 0.05). Alterations in the percentage of individual phospholipid classes were observed in both successive samples of BAL fluid when compared with those in the control subjects. PAF and PAF-AcH were detected in high levels on the first day (p < 0.001), which were reduced on the tenth day (p < 0.05). An inverse correlation between PAF levels and PaO2/FIO2 ratio was observed. Finally, the percentage of macrophages decreased and the percentage of neutrophils increased during the course of PTE. In conclusion, pulmonary embolism is associated with alterations in lung surfactant and inflammation in lung tissue, expressed by an increase in PAF and in neutrophils.

Bronchoalveolar lavage fluid characteristics of early intermediate and late phases of ARDS. Alterations in leukocytes, proteins, PAF and surfactant components.

OBJECTIVE: To determine the concentration of proteins and phospholipids, markers of inflammatory reaction such as platelet-activating factor (PAF), and cell alterations in bronchoalveolar lavage (BAL) fluid during the evolution of the acute respiratory distress syndrome (ARDS). DESIGN: Prospective controlled study. SETTING: 14-bed medical-surgical intensive care unit in a 750-bed university teaching hospital. PATIENTS: 19 mechanically ventilated patients, 9 patients with ARDS and 10 patients without cardiopulmonary disease (controls), were eligible for this study. INTERVENTIONS: BAL was performed during the early, intermediate, and late phases of ARDS. MEASUREMENTS AND RESULTS: Total phospholipids and individual phospholipid classes of the surfactant, proteins, PAF, and cells were measured. High levels of PAF, an increase in neutrophils and proteins, and quantitative as well as qualitative alterations in phospholipids in BAL fluid were observed in ARDS patients compared to the control group. PAF, proteins, and neutrophils were higher in early ARDS than in intermediate or late ARDS. The surfactant pool increased in the early phase and decreased in the intermediate or late phase of the syndrome. The qualitative alterations of surfactant consist of reduced phospholipid content in the surfactant structures with good surface properties; moreover, there was a considerable decrease in the percentage of phosphatidylcholine and phosphatidylglycerol, followed by an increase in phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin in all three phases of ARDS compared to the control group. Lysophosphatidylcholine was detectable only in late ARDS. CONCLUSION: Total surfactant phospholipids, surfactant components, and inflammatory markers such as PAF, cells, and proteins were affected in patients with ARDS. These factors, undergoing quantitative alterations during the course of ARDS, could have a significant role in the pathogenesis and evolution of ARDS.

Isolation, chemical characterization, and subcellular distribution of 1-O-alkyl-2-acetyl-sn-glycerol in Tetrahymena pyriformis cells.

1-O-Hexadecyl-2-acetyl-sn-glycerol, the immediate precursor of platelet- activating factor (PAF) in its de novo formation, was detected in the protozoon Tetrahymena pyriformis. It was purified from the total lipid extract by TLC, after successive developments in two different solvent systems. Characterization was assessed by (a) gas-liquid chromatography with electron capture detection, and (b) gas chromatography combined with mass spectrometry in selected ion monitoring mode, after derivatization with heptafluorobutyric acid anhydride and tert-butyldimethylchlorosilane/imidazole, respectively. Its quantity was found to be 0.1 nmol/10(7) cells from the GC-MS, using authentic alkylacetylglycerol as external standard. Cell fractionation revealed that alkylacetylglycerol is located exclusively in the microsomal fraction of the protozoon. Previously, we have reported the occurrence of PAF in the microsomal fraction, as well as a dithiothreitol-insensitive CDP-choline: cholinephosphotransferase activity that utilizes exogenous alkylacetylglycerol as substrate in the mitochondrial and microsomal fractions. The above findings indicate that PAF can be formed in the cell by the de novo pathway.

Studies on the subcellular distribution of 1-O-alkyl-2-acetyl-sn-glycero phosphocholine (PAF) and on the enzymic activities involved in its biosynthesis within the ciliate Tetrahymena pyriformis.

The ciliated protozoan Tetrahymena pyriformis contains platelet-activating factor (PAF) as a physiological minor lipid. Its subcellular localization was found as follows: 13.7% in the pellicles, 24.9% in mitochondria, 56.5% in microsomes and 7.1% in the cytosol. Succinate dehydrogenase was used as marker enzyme. PAF remains cell-associated unless bovine serum albumin is included in the extracellular medium. In this case 15% of total PAF, portion comparable to that found in the pellicles, is released. Investigation of the principal enzymic activities involved in PAF formation showed that PAF-acetyltransferase (2.3.167) is totally absent from the protozoan. This means that the 'remodelling' pathway occurring in pro-inflammatory cells does not contribute in PAF formation in our system. A dithiothreitol (DTT)-insensitive CDPcholine phosphocholinetransferase activity involved in PAF biosynthesis is shown for the first time to be responsible for PAF production in T. pyriformis. It uses exogenous alkyl-acetyl-glycerol as substrate and is saturated over substrate concentration 250 microM. It can also use endogenous lipids as substrate. It is distributed mainly in mitochondria and microsomes, much less is found in the pellicles and it is totally absent from the cytosol. Its insensitivity to DTT, its selectivity to alkyl-acetyl-G and its different distribution compared to the enzymic activity involved in PC formation (EC 2.7.8.2) suggest that a different enzyme, specific for PAF formation (EC2.7.8.16) via the de novo pathway exists in the protozoan.

A PAF-acetylhydrolase activity in Tetrahymena pyriformis cells.

Our study provides evidence for the existence of an acylhydrolase activity in Tetrahymena pyriformis cells, capable of hydrolyzing the sn-2 ester bond of the PAF molecule. This activity is mainly distributed in the microsomal fraction (76.5% of total) and has properties similar to the mammalian PAF-acetylhydrolase since it is Ca(2+)-independent, acid-labile, is inhibited by DFP and PMSF but it is not affected by egg yolk phosphatidylcholine. This microsomal acylhydrolase has apparent Km and Vmax values of 1.56 microM and 373 pmols.mg.min respectively. This is the first report of the existence of a PAF-acetylhydrolase activity in a non-mammalian cell.

In vivo metabolism of platelet-activating-factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by the protozoan Tetrahymena pyriformis.

The metabolism of exogenous platelet-activating-factor was studied in the protozoan Tetrahymena pyriformis in vivo. When the cells are exposed to 1.10(-6) M PAF, the molecule is rapidly metabolized to 1-O-alkyl-2-acyl(long chain)-GPC, a major component of the protozoan membranes. The appearance of lyso-PAF from the first minutes even in low levels provides evidence that deacetylation is an intermediate step. After incubation for 30 min, transformation to aminoethyl phosphonolipids is also observed. The fate of PAF in concentrations 1.5.10(-11) M or 1.10(-8) M PAF, was the same. An amount of PAF depending on the external PAF concentration remained intact in the cell even after 1 h incubation. Our results suggest that the easily cultured protozoan can be a useful model for studying PAF's metabolism.

Semisynthetic preparation of 1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor).

1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor, PAF), (1.9 mumol) was prepared from the total lipid extract of the protozoan Tetrahymena pyriformis 9 x 10(7) cells. The procedure involved mild alkaline hydrolysis of the total lipids, followed by acetylation and purification of the product by preparative TLC and HPLC. The yield was 60% with respect to the content of 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine in the total lipids, determined after preparative TLC. The alkyl side chain of the semisynthetic PAF was composed of hexadecyl residue. Our product was identified as PAF according to its biological activity, the chromatographic behaviour on TLC and HPLC, the physicochemical properties and the behaviour under treatment with PLA2 and Lipase from Rhizopus arrhizus. The above procedure is proposed as a facile, inexpensive and convenient method.