Renate Haupt

Preterm infants with high polyunsaturated fatty acid and plasmalogen content in tracheal aspirates develop bronchopulmonary dysplasia less often

Objective: Oxygen toxicity causes chronic bronchopulmonary dysplasia (BPD) in extremely preterm infants. Polyunsaturated fatty acids (PUFA) and plasmalogens are the two main substrates for lipid peroxidation in the pulmonary surfactant. In the present study, we tested whether low concentrations of both were associated with development of BPD and whether both were further reduced during mechanical ventilation with oxygen. Design: Prospective, noninterventional, descriptive study. Setting: Level III neonatal intensive care unit in a university hospital. Patients: In 25 extremely low birth weight infants with respiratory distress syndrome, tracheal aspirates were collected immediately after birth and in the following 4 days. As control, tracheal and pharyngeal aspirates were collected from healthy infants immediately after birth. The amount of PUFA and dimethylacetals (DMA, representing plasmalogens) was determined gaschromatographically. Interventions: None. Measurements and Main Results: The relative percentages of PUFA and DMA on all fatty acids in non‐BPD infants (PUFA% 26 ± 8.9, DMA% 3.5 ± 1.2) were higher compared with infants who developed BPD (PUFA% 14.5 ± 3.8, DMA% 1.8 ± 0.9). In term healthy infants, DMA% and PUFA% were in the same range as in the BPD group. The higher levels found for non‐BPD infants decreased after day 1 to values equal to the BPD group and remained low. Conclusions: The results suggest that initially higher levels of PUFA and plasmalogens in the tracheal effluent are associated with a reduced risk of developing BPD and are reduced during the first day of ventilation.

Fatty acid translocase/CD36 mediates the uptake of palmitate by type II pneumocytes

Type II pneumocytes, which synthesize, store, and secrete pulmonary surfactant, require exogenous fatty acids, in particular palmitic acid, for maximum surfactant synthesis. The uptake of palmitate by type II pneumocytes is thought to be protein mediated, but the protein involved has not been characterized. Here we show by RT-PCR and Northern blot analysis that rat type II pneumocytes express the mRNA for fatty acid translocase (FAT/CD36), a membrane-associated protein that is known to facilitate the uptake of fatty acids into adipocytes. The deduced amino acid sequence from rat type II pneumocytes reveals 98% identity to the FAT/CD36 sequence obtained from rat adipocytes. The uptake of palmitate by type II pneumocytes follows Michaelis-Menten kinetics (Michaelis-Menten constant = 11.9 ± 1.8 nM; maximum velocity = 62.7 ± 5.8 pmol ⋅ min-1 ⋅ 5 × 105pneumocytes-1) and decreases reversibly under conditions of ATP depletion to 35% of control uptake. Incubation of cells at 0°C inhibited the uptake of palmitate almost completely, whereas depletion of potassium was without effect. Preincubation of the cells with bromobimane or phloretin decreases the uptake of palmitate significantly as does preincubation with sulfo- N-succinimidyl oleate, the specific inhibitor of FAT/CD36 (C. M. Harmon, P. Luce, A. H. Beth, and N. A. Abumrad. J. Membr. Biol. 121: 261-268, 1991). From these data, we conclude that FAT/CD36 is expressed in type II pneumocytes and mediates the uptake of palmitate in a saturable and energy-dependent manner. The data suggest that the uptake process is independent of the formation of coated pits and endocytotic vesicles.Type II pneumocytes, which synthesize, store, and secrete pulmonary surfactant, require exogenous fatty acids, in particular palmitic acid, for maximum surfactant synthesis. The uptake of palmitate by type II pneumocytes is thought to be protein mediated, but the protein involved has not been characterized. Here we show by RT-PCR and Northern blot analysis that rat type II pneumocytes express the mRNA for fatty acid translocase (FAT/CD36), a membrane-associated protein that is known to facilitate the uptake of fatty acids into adipocytes. The deduced amino acid sequence from rat type II pneumocytes reveals 98% identity to the FAT/CD36 sequence obtained from rat adipocytes. The uptake of palmitate by type II pneumocytes follows Michaelis-Menten kinetics (Michaelis-Menten constant = 11.9 +/- 1.8 nM; maximum velocity = 62.7 +/- 5.8 pmol. min(-1). 5 x 10(5) pneumocytes(-1)) and decreases reversibly under conditions of ATP depletion to 35% of control uptake. Incubation of cells at 0 degrees C inhibited the uptake of palmitate almost completely, whereas depletion of potassium was without effect. Preincubation of the cells with bromobimane or phloretin decreases the uptake of palmitate significantly as does preincubation with sulfo-N-succinimidyl oleate, the specific inhibitor of FAT/CD36 (C. M. Harmon, P. Luce, A. H. Beth, and N. A. Abumrad. J. Membr. Biol. 121: 261-268, 1991). From these data, we conclude that FAT/CD36 is expressed in type II pneumocytes and mediates the uptake of palmitate in a saturable and energy-dependent manner. The data suggest that the uptake process is independent of the formation of coated pits and endocytotic vesicles.

Bioactive oxidized lipids in the plasma of cardiac surgical intensive care patients.

Critical illness is associated with increased oxidative stress that may give rise to the formation of lipid hydroperoxides (LOOH) and various secondary degradation products such as fragmented phosphatidylcholine (FPC) and lipids related to the platelet-activating factor (PAF). Because some oxidized phospholipids are potent proinflammatory agents, we measured the concentration of LOOH, FPC, and PAF-like activity in blood plasma of 36 patients who had undergone cardiac surgery and developed postoperative complications associated with systemic inflammatory response syndrome (SIRS) or multiple organ failure (MOF). These patients were compared to two control groups, namely preoperative patients scheduled for cardiac surgery (n = 13), and postoperative patients without complications (n = 19). Postoperative patients had higher concentrations of LOOH and lower concentrations of FPC than preoperative patients (P < 0.01). However, SIRS and MOF had no significant effect on the concentration of oxidatively modified lipids. This is despite the fact that MOF patients showed evidence of increased lipid peroxidation (7-fold higher ratio of &agr;-tocoquinone/&agr;-tocopherol compared to control). LOOH correlated positively with the white blood cell count. Postoperative patients had 4-fold higher plasma activities of phospholipase A2 and this activity was further increased in patients with SIRS (P < 0.04). Phospholipase A2 activity correlated negatively with the concentration of FPC. The data suggest that oxidatively modified lipids do not accumulate in patients with SIRS and MOF, perhaps because enhanced peroxidation of lipids is offset by enhanced lipolytic activity.

Study of platelet-activating factor acetylhydrolase in the perioperative period of patients undergoing cardiac surgery

ABSTRACT After operations with cardiopulmonary bypass, patients often show early symptoms of the systemic inflammatory response syndrome (SIRS). Potential mediators of SIRS include the platelet-activating factor (PAF), which has been linked to septic shock and multiple organ dysfunction. We studied the effect of cardiac surgery on PAF acetylhydrolase, the PAF-degrading plasma enzyme, as well as the relationship between the enzyme and the postoperative state of the patients. PAF acetylhydrolase activity decreased by 38 ± 8% after instituting cardiopulmonary bypass because of plasma dilution and returned to near-preoperative levels within 6 h postsurgery. After that, enzyme levels decreased again, resulting in a 24 ± 12% reduction until at least 3 days postsurgery. Patients in poor postoperative condition (Acute Physiology Score >9) had a lower preoperative PAF acetylhydrolase activity than did normal patients (12 ± 4 vs. 17 ± 4 nmol min-1 mL-1; p < .05). Likewise, patients who developed postoperative SIRS had a lower preoperative PAF acetylhydrolase activity than did patients without SIRS (12 ± 3 vs. 17 ± 4 nmol min-1 mL-1; p < .05). The data suggest that PAF acetylhydrolase deficiency is among the factors associated with postoperative distress after cardiac surgery.

Low molecular mass trypsin inhibitors in normal and osteoarthritic human articular cartilage

A modified radial diffusion assay was used for the direct semiquantitative determination of low molecular mass trypsin inhibitors in small samples of human cartilage. The low molecular mass trypsin inhibitor in articular cartilage of normal human femoral heads is not distributed evenly but occurs in areas of low, medium and high content. The weight-bearing area of the femoral head belongs among the regions with low inhibitor content. The results obtained with osteoarthritic femoral heads showed that the inhibitor content in osteoarthritic cartilage is significantly lower than that in normal articular cartilage (p less than 0.1%).

Are Biochemical Parameters of Lung Maturation from Gastric, Tracheal, and Pharyngeal Aspirates Comparable?

Phospholipid parameters – lecithin/sphingomyelin (L/S) ratio and palmitic acid/stearic acid (P/S) ratio – of gastric (GA), pharyngeal (PA), and tracheal aspirates (TA) have often been used for estimation of lung maturation. However, it was not known whether values from different sources can be alternatively used. The study was performed to test whether P/S and L/S ratios in GA, PA, and TA are comparable. 11 GA, 30 PA, and 10 TA were obtained from infants within 10 min after birth. All infants were healthy, vaginally delivered with no respiratory distress, and had a gestational age ≥34 weeks. Patterns of fatty acids were determined by gas chromatography and the L/S ratio by high-performance liquid chromatography. We found lower L/S ratios in GA (16.5 ± 5.2) when compared with PA (39 ± 16) or TA (41 ± 15; p < 0.01); there were no differences between PA and TA. The P/S ratio was in the same range in PA and TA, but lower in GA. As compared with TA and PA, in GA the relative percentage of palmitic acid of all fatty acids was lower, the percentage of monounsaturated fatty acids, however, higher. We conclude that phospholipid parameters of lung maturation in PA and TA, but not in GA, can be alternatively used.

Semiquantitative determination of low molecular weight proteinase inhibitors in the presence of high molecular weight inhibitors by a modified radial diffusion assay.

The radial diffusion assay is very suitable for the determination of proteinase inhibitors in biological fluids. By combining radial diffusion and ultrafiltration, it has become possible to directly determine low molecular weight proteinase inhibitors in mixtures with inhibitors of higher molecular weight. By this modification the inhibitor solutions to be investigated are not pipetted into wells as usually, but are applied on small pieces of dialysis membranes lying on the gel. The exclusion limit of the membrane must be of a magnitude that the inhibitors of higher molecular weight are retained, whereas the inhibitors of lower molecular weight can diffuse into the gel. The modified method can be used for the direct determination of e.g. aprotinin (Mr 6500) in the presence of alpha 1-proteinase inhibitor (Mr 54,000), ovoinhibitor (Mr 50,000) and ovomucoid (Mr 27,000), respectively. The modified method is suitable for the direct determination of low molecular weight inhibitors of trypsin and papain in serum, synovial fluid and saliva. Tissue extracts containing 4 M guanidine hydrochloride or 6 M urea can be investigated directly, too.

THE ANTIOXIDATIVE POTENTIAL OF ALVEOLAR SURFACTANT

Alveolar surfactant is exposed to a variety of oxidants that can oxidize functionally important lipids and proteins. We examined the hypothesis that the type II pneumocyte equips surfactant with antioxidants to counteract its oxidation.Rat type II cells, cultured in the presence of 14C-palmitic acid and either 3H-vitamin E or 3H-vitamin D, responded to stimulation with isoproterenol with a time-dependent increase in secretion of 14C-labelled phospholipids and 3H-vitamin E, but not of 3H-vitamin D.Plasmatogens - a subclass of phospholipids - also act as antioxidants in animal cells. Type II cells, cultured in presence of 3H palmitic acid and 14C hexadecanol, synthesize and secrete 3H labeled phospholipids and 14C labeled plasmalogens spontaneously and in response to isoproterenol stimulation.In pharyngeal aspirates of healthy newborns vitamin Eand plasmalogen contents range from 2-10 nmol/μmol polyunsaturated fatty acids (PUFA) and from 8-20 nmol/μmol PUFA respectively.We conclude that alveolar surfactant is equipped with lipophilic antioxidants of its own during its formation in type II cells. These lipophilic components could be of use as clinical parameters to evaluate the antioxidantive potential of alveolar surfactant.Supported by BMFT Project “Risikoneugeborenes”.