B. Moseley Waite

A colorimetric method for amniotic fluid phospholipids and their relationship to the respiratory distress syndrome

Abstract A recently published work has stated that the amniotic fluid lecithin concentration is an accurate method of predicting the development of the respiratory distress syndrome (RDS). We have developed a simpler method for estimating amniotic fluid phospholipids, and 204 amniotic fluids from 120 pregnancies were studied. The level of hydrolyzable phospholibids (of which lecithin represents approximately 70 per cent in the third trimester) rises in the third trimester, but a signalling surge is not seen in compiled data. Four of five cases in which RDS did occur were found to have the expected low level of amniotic fluid hydrolyzable phospholipid just prior to birth. However, RDS did not occur in a large group of infants whose amniotic fluid hydrolyzable phospholipid concentrations were low. We, therefore, believe that, though this method would be reasonably valid as a determinant of time of delivery if high levels were found, low levels are not necessarily indicative that RDS will occur.

The acyl-CoA desaturases of microsomes from rat liver and the Morris 7777 hepatoma

We have investigated the role of the microsomal oxidative desaturase in defining the aberrant phosphoglyceride fatty acid composition of hepatomas. The microsomal delta 9-stearoyl-CoA, delta 6-oleoyl(linolenoyl)-CoA, and delta 5-eicosatrienoyl-CA desaturase activities were studied in control and host liver and in the poorly differentiated Morris 7777 hepatoma. The delta 9-stearoyl-CoA desaturase of the hepatoma was significantly decreased (42%) relative to control liver, yet the hepatoma specific activity was twice that of host liver. Additionally, the specific activity of the delta 9-stearoyl-CoA desaturase of the tumor was found to decrease with increasing tumor weight. Also this desaturase was inactivated by freezing and thawing. The delta 6-oleoyl(linolenoyl)-CoA and delta 5-eicosatrienoyl-CoA desaturases of the hepatoma were 39% and 4% of control, respectively. The electron transport components involved in the desaturase system were reduced, although this did not appear to be rate-limiting. In addition, two competing metabolic reactions which could lower the observed desaturase activities, hydrolysis of the thioester and incorporation of substrate acyl-CoA molecules into glycerides, did not appear to be responsible for the lowered desaturase activities of the tumor. Thus, it appears that reduced levels of the desaturases themselves may be responsible for the observed activities. These results indicate that the capacity of the hepatoma to biosynthesize polyunsaturated fatty acids is greatly reduced and this is consistent with the decreased polyene content observed in many neoplasms.

Lyso(bis)phosphatidic acid: a novel source of arachidonic acid for oxidative metabolism by rabbit alveolar macrophages.

To identify the source of arachidonic acid utilized for eicosanoid production, rabbit alveolar macrophages were challenged with 1.0 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) or 3.8 microM Ca+2 ionophore A23187 for 3 h. Upon stimulation with TPA, a loss of [3H]arachidonic acid from phosphatidylcholine, phosphatidylethanolamine, lyso(bis)phosphatidic acid, and phosphatidylserine/phosphatidylinositol was observed. Although calcium ionophore stimulated the liberation of arachidonate solely from phosphatidyl-ethanolamine and phosphatidylcholine, it proved to be a poor stimulus for macrophage-synthesis of eicosanoids. Our evidence suggests that degradation of phosphatidylinositol and lyso(bis)phosphatidic acid induced by TPA yields a source of arachidonate which is the preferred substrate for oxidative metabolism by the cyclooxygenase and lipoxygenase pathways.

Surfactant protein B inhibits secretory phospholipase A2 hydrolysis of surfactant phospholipids

Hydrolysis of surfactant phospholipids (PL) by secretory phospholipases A(2) (sPLA(2)) contributes to surfactant damage in inflammatory airway diseases such as acute lung injury/acute respiratory distress syndrome. We and others have reported that each sPLA(2) exhibits specificity in hydrolyzing different PLs in pulmonary surfactant and that the presence of hydrophilic surfactant protein A (SP-A) alters sPLA(2)-mediated hydrolysis. This report tests the hypothesis that hydrophobic SP-B also inhibits sPLA(2)-mediated surfactant hydrolysis. Three surfactant preparations were used containing varied amounts of SP-B and radiolabeled tracers of phosphatidylcholine (PC) or phosphatidylglycerol (PG): 1) washed ovine surfactant (OS) (pre- and postorganic extraction) compared with Survanta (protein poor), 2) Survanta supplemented with purified bovine SP-B (1-5%, wt/wt), and 3) a mixture of dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (DPPC:POPC:POPG, 40:40:20) prepared as vesicles and monomolecular films in the presence or absence of SP-B. Hydrolysis of PG and PC by Group IB sPLA(2) (PLA2G1A) was significantly lower in the extracted OS, which contains SP-B, compared with Survanta (P = 0.005), which is SP-B poor. Hydrolysis of PG and PC in nonextracted OS, which contains all SPs, was lower than both Survanta and extracted OS. When Survanta was supplemented with 1% SP-B, PG and PC hydrolysis by PLA2G1B was significantly lower (P < 0.001) than in Survanta alone. When supplemented into pure lipid vesicles and monomolecular films composed of PG and PC mixtures, SP-B also inhibited hydrolysis by both PLA2G1B and Group IIA sPLA2 (PLA2G2A). In films, PLA2G1B hydrolyzed surfactant PL monolayers at surface pressures ≤30 mN/m (P < 0.01), and SP-B lowered the surface pressure range at which hydrolysis can occur. These results suggest the hydrophobic SP, SP-B, protects alveolar surfactant PL from hydrolysis mediated by multiple sPLA(2) in both vesicles (alveolar subphase) and monomolecular films (air-liquid interface).

Phospholipase A activities in rat placentas of 14 days' gestation.

1. Homogenates and subcellular fractions of placentas obtained from rats on the 14th day of gestation were assayed for enzymes characteristic of various subcellular organelles. Based on these assays it seemed the placental lysosomes (acid phosphatase activity) were distributed equally among the subcellular particulate fractions and that the cytosol was not contaminated by mitochondria or microsomes. 2. The placental preparations were assayed for phospholipase A activity using 2-[14-C] phosphatidylethanolamine. We found phospholipases A with pH optima of 4.0, 7.0, and 8.5. These enzymes had Ca-2+ requirements and subcellular localizations similar to those reported for adult rat liver (Waite, M., Scherphof, G.L., Boshouivers, F.M.G. and van Deenen, L.L.M. (1969) J. Lipid Res. 10, 411.) 3. The majority of the placental phospholipase A activity was found in the cytosol. We think this activity represents enzyme(s) solubilized from the plasma membranes of placental cells and speculate that this enzyme may be active in a placental transport mechanism.

CO-LOCALIZATION OF SUPEROXIDE GENERATION AND NADP FORMATION IN PLASMA MEMBRANE FRACTIONS FROM HUMAN NEUTROPHILS ~

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for piasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, andΒ-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.

Cholinephosphotransferase activities in early rat embryos and their associated placentas

Abstract CDP-Choline:1,2-diglycerolcholinephosphotransferase (EC 2.7.8.2, cholinephosphotransferase) activities were determined in subcellular fractions prepared from rat embryos, placentas, or yolk sacs obtained on the fourteenth day of gestation. It was found that, in all of the tissues studied, cholinephosphotransferase activity (1) copurified with NADPH-cytochrome c reductase activity (EC 1.6.2.4), (2) was maximal around pH 8.0; (3) was stimulated by MgCl2, exogenous diolein, and cytidine diphosphocholine (CDP-choline); and (4) was highest in homogenates of placentas, lowest in those of embryos, and intermediate in those of yolk sacs. These data substantiate, for the first time, that the early mammalian (rat) embryo, placenta, and yolk sac have the ability to synthesize phospholipids de novo.