Lower Phospholipid Content Research Articles

Dilute prothrombin time-based lupus ratio test: Integrated LA testing with recombinant tissue thromboplastin

The lupus ratio (LR) test is a normalized ratio of the clotting times obtained with low and high phospholipid (PL) concentrations, where the test plasma is mixed 1:1 with normal pooled plasma (NP). As an integrated, automated, and computer-assisted assay, this principle has been applied to the dilute activated partial thromboplastin time (dAPTT) and dilute Russell viper venom time (dRVVT) test systems. In this study, we used recombinant thromboplastin to develop an automated LR test based on the dilute prothrombin time (dPT). Using plasma samples from a selected group of patients ( N=92) with a well-defined lupus anticoagulant (LA) status, the dPT-based LR test showed fair agreement with the dAPTT-based LR test ( κ=.60, P<.001) and good agreement with the dRVVT-based LR test ( κ=.78, P<.001) regarding the classification of plasmas as LA-negative or -positive. Most discordant plasmas were low positive. Correlation between dPT- and dRVVT-positive LRs was only moderate ( r s=.51, P=.002), and correlation between dPT- and dAPTT-positive LRs was nonsignificant ( r s=.23, P=.18). Using dilutions of pooled LA-positive plasma, the dAPTT-based LR test appeared to be the most sensitive of the three tests. This study confirms the general validity of the LR principle and shows that the dPT-based LR test yields reproducible results. The low coefficients of variation (CV) of the LR assays, combined with a fairly low correlation among the three tests, support the assumption of antibody heterogeneity of LA-positive plasmas and the desirability of performing more than one test.

Antioxidant enzymes and paraoxonase show a co-activity in preserving low-density lipoprotein from oxidation.

Oxidative modification of low-density lipoprotein in the artery wall plays a crucial role in the development of atherosclerosis. This physiopathological mechanism is clearly inhibited by high-density lipoprotein possibly via paraoxonase enzyme activity, present in high-density lipoprotein. In this study, we determined the in vitro susceptibility of low-density lipoprotein to oxidation and the effect of various factors, such as paraoxonase phenotypes, on this process. Low-density lipoprotein from healthy volunteers (n=66) was isolated using the precipitant reagent and the oxidation was evaluated by measuring the malonyl dialdehyde and diene levels. Low-density lipoprotein cholesterol and phospholipid, vitamin E, serum cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol levels, and erythrocyte antioxidant enzymes were also determined. There was no difference among the parameters with regard to gender. Low-density lipoprotein samples obtained from subjects with the AA allele were more prone to oxidation, as observed by their higher stimulated conjugated diene (P=0.041) and thiobarbituric acid-related substance (P=0.042) levels, than samples from subjects with AB or BB alleles. The subjects with the BB allele had higher superoxide dismutase (P=0.021) and catalase (insignificant increase) activities, while their conjugated diene (P=0.000) levels were lower. In conclusion, our results revealed that the high low-density lipoprotein oxidation is related to the high low-density lipoprotein cholesterol content and low phospholipid content. The present study demonstrated an increase in superoxide dismutase and catalase activities, as well as PON1 activities, in subjects with the BB allele. Since these enzymes all show activity against low-density lipoprotein oxidation, we propose that future investigations on atherosclerotic processes should address PON1 polymorphism as well as PON1 and other antioxidant enzymes.

The Evaluation of Clotting Times in the Laboratory Detection of Lupus Anticoagulants

Although there is international consensus regarding the general principles of testing for lupus anticoagulants (LAs), no agreement exists as far as the analysis of the clotting time results is concerned. Twenty-nine laboratories participating in the Fifth International Survey of Lupus Anticoagulants (ISLA-5) reported the activated partial thromboplastin time (APTT)-based clotting times obtained on seven defined test samples and a normal plasma (NP) using the same two reagents with low and high phospholipid (PL) concentrations, respectively. These clotting times were used to analyse how various methods of calculating the results may influence the apparent sensitivity of LA tests. We found that the use of a separate screening test may lead to the exclusion of samples where the presence of LA would have been detected by a combined screening and confirmatory method. For instance, the dilute APTT (dAPTT) gave a sensitivity of 53.5% (screening test), while the calculation of a ratio between the clotting times obtained with two different PL concentrations gave a sensitivity of 68.1% (confirmatory test). The normalisation of results by dividing with the corresponding results of NP increased the apparent sensitivity. The screening test ratio between dAPTT results of test samples and NP gave a sensitivity of 84.7%. The normalised ratio between the clotting times obtained with the two reagents (lupus ratio, LR) gave a sensitivity of 95.1%. We conclude that when testing for LA, all samples should be tested with both low (screening procedure) and high (confirmatory procedure) PL concentrations. These two clotting times should be evaluated in relation to each other and to the corresponding results obtained with a reference plasma (normalisation).

Phospholipids modulate the substrate specificity of soluble UDP-glucose:steroid glucosyltransferase from eggplant leaves

UDP-glucose-dependent glucosylation of solasodine and diosgenin by a soluble, partially purified enzyme fraction from eggplant leaves is affected in a markedly different way by some phospholipids. While glucosylation of diosgenin and some closely related spirostanols, e.g. tigogenin or yamogenin, is strongly inhibited by relatively low concentrations of several phospholipids, the glucosylation of solasodine is unaffected or even slightly stimulated. These effects depend both on the structure of the polar head group and the nature of the acyl chains present in the phospholipid. The most potent inhibitors of diosgenin glucosylation are choline-containing lipids: phosphatidylcholine (PC) and sphingomyelin (SM) but the removal of phosphocholine moiety from these phospholipids by treatment with phospholipase C results in an almost complete recovery of the diosgenin glucoside formation by the enzyme. Significant inhibition of diosgenin glucoside synthesis and stimulation of solasodine glucosylation was found only with PC molecular species containing fatty acids with chain length of 12–18 carbon atoms. PC with shorter or longer acyl chains had little effect on glucosylation of either diosgenin or solasodine. Our results indicate that interaction between the investigated glucosyltransferase and lipids are quite specific and suggest that modulation of the enzyme activity by the nature of the lipid environment may be of importance for regulation of in vivo synthesis of steroidal saponins and glycoalkaloids in eggplant.

Phosphorus status affects postharvest respiration, membrane permeability and lipid chemistry of European seedless cucumber fruit (Cucumis sativus L.)

Fruit of European seedless cucumber were grown in a greenhouse under low and high phosphorus (P) fertilization regimes. Tissue P concentration of fruit (number one grade) from low-P plants was, on average, 45% of that of fruit from high-P plants. Fruit P status affected membrane lipid chemistry and fruit respiration after harvest. Mesocarp tissue of low-P fruit had a lower concentration of phospholipids, lower level of unsaturation in various pools of fatty acids, and a greater rate of electrolyte leakage than that of high-P fruit. On average, respiration of low-P fruit was 21% higher than that of high-P fruit over a 16-day postharvest interval at 23°C. Moreover, low-P fruit experienced a climacteric in respiration that began about 40 h after harvest, reached a maximum at 72 h, and declined to preclimacteric levels by 90 h. The difference in respiration rate between low- and high-P fruit was as high as 57% during the climacteric. The respiratory climacteric was unique to the low-P fruit and was not associated with an increase in fruit ethylene concentration or ripening. Phosphorus nutrition can thus alter the postharvest physiology of cucumber fruit by affecting membrane lipid chemistry, membrane integrity and respiratory metabolism.

Long-term effects of cis and trans monounsaturated (18:1) and saturated (16:0) fatty acids on the synthesis and secretion of apolipoprotein A-I- and apolipoprotein B-containing lipoproteins in HepG2 cells

The objective of this study was to compare the long-term effects of oleic (cis 18:1), elaidic (trans 18:1), and palmitic (16:0) acids on hepatic lipoprotein production, using HepG2 cells as an experimental model. The net accumulation in the medium of apolipoprotein A-I (apoA-I) was not significantly altered by fatty acids, whereas that of apoB was increased with oleic and elaidic acids. Oleic acid, and to a lesser extent elaidic and palmitic acids, increased the mass of triglycerides in the medium and the incorporation of [(3)H]glycerol into secreted triglycerides. The incorporation of [(14)C]acetate into cellular and secreted total cholesterol was stimulated by 96% and 83%, respectively, with elaidic acid but was not significantly modified by oleic or palmitic acid. Relative to oleic acid, the secretion of (14)C-labeled phospholipids and triglycerides was decreased 28% to 31% with elaidic and palmitic acids whereas that of free cholesterol and cholesteryl esters was enhanced 93% and 73%, respectively, with elaidic acid but remained unchanged with palmitic acid. Compared with oleic acid, elaidic acid stimulated the secretion of very low density lipoprotein cholesterol (VLDL-Chol), low density lipoprotein cholesterol (LDL-Chol), and high density lipoprotein cholesterol (HDL-Chol) by 43%, 70%, and 34%, respectively, whereas palmitic acid decreased VLDL-Chol but had no significant effect on LDL-Chol and HDL-Chol. The ratios of total cholesterol to HDL-Chol were 3.17, 3.60, and 3.25 with oleic, elaidic, and palmitic acids, respectively; the corresponding ratios of LDL-Chol to HDL-Chol were 0.87, 1.10, and 0.93, respectively. Compared with oleic and palmitic acids, the LDL and HDL particles secreted in the presence of elaidic acid contained higher levels of free cholesterol and cholesteryl esters and a lower content of phospholipids. The phospholipid-to-total cholesterol ratios of HDL were 1.05, 0.40, and 0.76 with oleic, elaidic, and palmitic acids, respectively. Our results indicate that in comparison with cis monounsaturated and saturated fatty acids, trans fatty acids have more adverse effects on the concentration and composition of lipoproteins secreted by HepG2 cells.

The Lupus Ratio Test – An Interlaboratory Study on the Detection of Lupus Anticoagulants by an APTT-based, Integrated, and Semi-quantitative Test

The Lupus Ratio (LR) test for lupus anticoagulants integrates screening, mixing with normal plasma and confirmation procedures into one assay. The sensitivity and reproducibility of the APTT based version of this assay was tested in an interlaboratory study that was part of the Fifth International Survey of Lupus Anticoagulants (ISLA-5). One LA negative plasma containing heparin, six LA positive plasmas and a normal pooled plasma (NP) were distributed to 31 laboratories world-wide together with two APTT reagents, one with a high and one with a low phospholipid concentration. The laboratories performed two APTTs, one with each reagent, on 1:1 mixtures of test plasma and NP. The ratio between the two clotting times was divided by the corresponding ratio for the NP. This final ratio is the LR of that plasma. The overall sensitivity was found to be 95.1%, and the normal, heparin-containing sample was reported to be negative by all the laboratories. When the results were grouped in low, medium and high positive plasmas, a "consensus" regarding the strength of each plasma was easily found. 85.0% of the results were in agreement with this consensus. This study shows that with the LR test, it is possible to obtain high interlaboratory agreement regarding the presence or absence of LA as well as the semi-quantification of this inhibitor.

Regulation of Prothrombinase Activity by Protein S

The independent effect of protein S as prothrombinase inhibitor has been proposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrations of protein S (300 nM) equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low phospholipid concentrations (approximately 0.2 microM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd approximately 10(-8) M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin generation rates at low phospholipid in the presence of protein S based on the diminished available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the phospholipid prior to the addition of factor Xa, factor Va and prothrombin. The results indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course of the physiological thrombin generation. The effect of protein S was therefore studied on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation when surface is provided at physiologic concentrations using isolated platelets (2 x 10(8)/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effects of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generation, indicate that protein S and protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.

Lupus Anticoagulant Testing in Europe: An Analysis of Results from the First European Concerted Action on Thrombophilia (ECAT) Survey Using Plasmas Spiked with Monoclonal Antibodies against Human β2-Glycoprotein I

Lupus anticoagulants (LA) are immunoglobulins directed to either prothrombin or Beta-2-glycoprotein 1(beta1GPI) bound to phospholipids. Most patients with LA have both beta2GPI- and prothrombin-dependent antibodies. Several recent reports have shown that LA is more strongly associated with thrombosis than anticardiolipin antibodies (aCL). Therefore, an accurate detection of LA is of utmost importance in patients suspected of an antiphospholipid syndrome. We recently raised a series of murine monoclonal antibodies against human Beta-2-glycoprotein I (beta2GPI) with LA activity similar to affinity purified human beta2GPI-dependent LAs. A normal plasma pool, and the same pool spiked with LA positive anti-beta2GPI antibodies at two potency levels, were used as materials in an external quality assessment scheme organised by the European Concerted Action on Thrombosis (ECAT). Fifty nine laboratories participating in this trial were asked to test for the presence of a LA in the 3 samples submitted. The majority (82%) of the participants found the high potency LA sample to be positive. Only 37% of the laboratories considered the weak potency LA sample to be positive. The submission of a normal sample, a weakly positive sample and a clearly positive sample enabled us to compare the relative LA responsiveness of the different screening assays used. Clotting time ratios varied from 0.81 to 3.28 for sample B and from 0.66 to 5.32 for sample D. In general, the highest clotting time ratios were found with the dilute prothrombin time (dPT), the dilute Russell Viper Venom time (dRVVT) and the Kaolin Clotting time. The most frequently used screening tests were the aPTT and the dRVVT. With the various assay systems, LA responsiveness varied largely according to the reagents used. For the beta2GPI-dependent LA used in this study, PTT LA clearly showed the highest responsiveness among the aPTT reagents and Innovin among the dPT reagents. The present study also shows that many laboratories still rely on poorly responsive screening assays for their LA tests. Other laboratories rely on sensitive and more specific integrated test systems based on a sensitive screening assay with a low phospholipid content and a confirmatory test employing high phospholipid concentrations. The most used integrated system was dRVVT based. However, also here the LA responsiveness was largely reagent dependent. In conclusion, many laboratories still rely on poorly responsive screening assays by which weakly positive LA samples are misdiagnosed. LA positive anti-beta2GPI moabs have a potential for the unlimited production of LA control specimens, that may help hemostasis laboratories choose more LA responsive assay systems and to assess intralaboratory precision of their LA testing.

Enhancement of Human Protein C Function by Site-directed Mutagenesis of the γ-Carboxyglutamic Acid Domain

This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-11 is unique to human protein C, whereas all other vitamin K-dependent proteins contain glycine. This site is located in a compact region of the protein, close to a suggested membrane contact site. Additional changes of H10Q or S12N resulted in proteins with lower calcium requirement for membrane contact but without further increase in membrane affinity at saturating calcium. Mutations Q32E and N33D did not, by themselves, alter membrane affinity to a significant degree. These mutations were included in other mutant proteins and may contribute somewhat to higher function in these mutants. This family of mutants helped discriminate events that are necessary for protein-membrane binding. These include calcium binding to the free protein and subsequent protein-membrane contact. Depending on conditions of the assay used, the mutants displayed increased activity of the corresponding activated protein C (APC) derivatives. The degree of enhanced activity (up to 10-fold) was dependent on the concentration of phospholipid and quality of phospholipid (+/- phosphatidylethanolamine) used in the assay. This was expected, because APC is active in its membrane-associated form, which can be regulated by changes in either the protein or phospholipid. As expected, the largest impact of the mutants occurred at low phospholipid concentration and in the absence of phosphatidylethanolamine. The anticoagulant activity of all proteins was stimulated by protein S, with the greatest impact on the enhanced mutants. Whereas plasma containing Factor V:R506Q was partially resistant to all forms of APC, the enhanced variants were more active than normal APC. Protein C variants with enhanced function present new reagents for study of coagulation and may offer improved materials for biomedical applications.

The aminosterol antibiotic squalamine permeabilizes large unilamellar phospholipid vesicles

The ability of the shark antimicrobial aminosterol squalamine to induce the leakage of polar fluorescent dyes from large unilamellar phospholipid vesicles (LUVs) has been measured. Micromolar squalamine causes leakage of carboxyfluorescein (CF) from vesicles prepared from the anionic phospholipids phosphatidylglycerol (PG), phosphatidylserine (PS), and cardiolipin. Binding analyses based on the leakage data show that squalamine has its highest affinity to phosphatidylglycerol membranes, followed by phosphatidylserine and cardiolipin membranes. Squalamine will also induce the leakage of CF from phosphatidylcholine (PC) LUVs at low phospholipid concentrations. At high phospholipid concentrations, the leakage of CF from PC LUVs deviates from a simple dose–response relationship, and it appears that some of the squalamine can no longer cause leakage. Fluorescent dye leakage generated by squalamine is graded, suggesting the formation of a discrete membrane pore rather than a generalized disruption of vesicular membranes. By using fluorescently labeled dextrans of different molecular weight, material with molecular weight ≤4000 g/mol is released from vesicles by squalamine, but material with molecular weight ≥10,000 is retained. Negative stain electron microscopy of squalamine-treated LUVs shows that squalamine decreases the average vesicular size in a concentration-dependent manner. Squalamine decreases the size of vesicles containing anionic phospholipid at a lower squalamine/lipid molar ratio than pure PC LUVs. In a centrifugation assay, squalamine solubilizes phospholipid, but only at significantly higher squalamine/phospholipid ratios than required for either dye leakage or vesicle size reduction. Squalamine solubilizes PC at lower squalamine/phospholipid ratios than PG. We suggest that squalamine complexes with phospholipid to form a discrete structure within the bilayers of LUVs, resulting in the transient leakage of small encapsulated molecules. At higher squalamine/phospholipid ratios, these structures release from the bilayers and aggregate to form either new vesicles or squalamine/phospholipid mixed micelles.

Aging of Soya-PC Liposomes Containing Vitamin E Reverses the Stimulating Effects of Freshly Prepared Liposomes on Growth of Fibroblasts in Culture

Extruded soya-PC liposomes (VET) averaging 80.4 ± 24.3 nm in diameter and containing 0 to 25% vitamin E were added to human fibroblasts in culture immediately after preparation or after a 4 month cold storage period (aged liposomes). Following 8 days of incubation, the effect on the growth, membrane permeability, and protein content of the fibroblasts was determined. The freshly prepared liposomes induced a proliferative effect on cell growth at low phospholipid concentrations (10 and 50μM). This effect was lost as the phospholipid concentration increased, and at concentrations exceeding 100 μM the toxic effects of liposomes became apparent. The incorporation of vitamin E into the liposomes reduced this toxicity. Aged liposomes showed a loss of proliferative activity at phospholipid levels of 10 and 50 μM. The age of the liposomes also influenced the protective effect of vitamin E on the cultures. Liposomes containing the vitamin and stored prior to use showed no proliferative effect, and cell toxicity increased with the percentage of vitamin initially present in the liposomes. The results suggest that vitamin E, incorporated into freshly prepared liposomes, is able to protect fibroblasts in culture from the toxic effects shown by phospholipid concentrations above 100 μM. This protective effect was lost when liposomes were stored prior to use.

Human gallbladder mucosal function: effects on intraluminal fluid and lipid composition in health and disease.

Gallbladder mucosal absorption of fluid during fasting is a well-known process. Indirect in vivo and recent in vitro evidence for physiologically relevant gallbladder absorption of cholesterol and phospholipids from bile has been observed in humans. The present study explored and compared by indirect means the relative efficiences of human gallbladder mucosal absorption of fluid and lipids in health and disease. Biliary lipids and pigment content were measured in fasting gallbladder bile samples obtained from gallstone-free controls and from four study groups: multiple and solitary cholesterol gallstone patients, and morbidly obese subjects with and without gallstones. Bile salts and pigment content were significantly greater in gallstone-free controls than in all other disease study groups. This was interpreted as evidence of more effective gallbladder mucosal fluid absorption in nonobese gallstone-free controls compared to that in all other groups. Correlation plot analyses of biliary lipids showed lower concentrations of phospholipids than expected from the index bile salt concentrations. The same was found for cholesterol concentrations but only in supersaturated samples. These findings were much more pronounced in gallstone free-controls and were accordingly interpreted as evidence of more efficient gallbladder absorption of both phospholipids and cholesterol in controls compared with that found in each of the disease study groups. Moreover, impaired gallbladder mucosal function, while invariably associated with cholesterol gallstone disease, was not found to be a necessary consequence of the physical presence of stones. It is concluded that efficient gallbladder mucosal absorption of both fluid and apolar lipids from bile is a normal physiological process that is often seriously impaired in the presence of either cholesterol gallstone disease or at least one of its precursor forms.

Membrane-binding properties of phospholipase C-beta1 and phospholipaseC-beta2: role of the C-terminus and effects of polyphosphoinositides, G-proteins and Ca2+.

We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-beta1 and -beta2, to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10(-3) M. PLC-beta1 and PLC-beta2 bind vesicles composed of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (molar ratio 1:1:1) with an approximate Kd of 10(-5) M. Inclusion of 2% PtdIns(4,5)P2 in these vesicles had no effect on the affinity of this interaction. As reported by others, removal of the C-terminus of PLC-beta1 and PLC-beta2 produces catalytically active fragments. The affinity of these truncated proteins for phospholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inclusion of G-protein alpha- and betagamma-subunit activators in the phospholipid vesicles does not increase the binding of PLC-beta1 or PLC-beta2, and the magnitude of G-protein-mediated PLC activation observed at low phospholipid concentrations (10(-6) M) is comparable to that observed at concentrations at which the enzymes are predominantly membrane-bound (10(-3) M). PLC-beta1 and -beta2 contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicate that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-proteins results from a regulated interaction between the membrane-bound proteins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface.

Cell lysis and release of particulate polysaccharides in extensive marine mucilage assessed by lipid biomarkers and molecular probes

During the massive mucllage event in the northern Adriatic Sea in July 1991 samples of macroaggregate were fixed in different ways: with formaldehyde, deep frozen and freeze-dried. Conventional microscopy (light and epifluorescence) revealed different autotrophic species embedded in gelatinous matl-ix. Cyanobacteria and heterotrophic bacteria were also identified. Scanning confocal laser microscopy (SCLMI and fluorescent molec~~ la r p obes (the lectins concanavalin A and UEA-I) showed wall-free cytoplasm and particulate polysacchar~des leaklng from the envelopes of broken cells in the matrix. The extensive cell lysis was supported by the observation of cytoplasn1-free cytoskeletons, stained by the molecular probe phalloidin High concentrations of triglycerides (30Y0 of total lipids) and free fatty aclds (22'2%) along with very low concentrations of phospholipids ( 2 % ) also indicated massive cell degradation in freeze-dried material. The mucllage observations were compared with those of a natural plankton community grown under hlgh nutrlent conditions using the same techniques. Free polysaccharides were observed as globular flocs (marine snow) during in situ enrichment experiments and inti-acellular polysaccharides as carbon storage materials In autotrophic organisms. No strings, filaments, layers, cell lysis or lipid classes indicating strong cell biodeterioration were observed in a 1 mo controlled experiment during an algal bloom.

Mobilisierbarkeit von hydrophoben organischen Schadstoffen in belasteten Böden und Abfällen. Teil I: Mobilisierbarkeit von PCB, PAK und n‐Alkanen durch Lösungsvermittler

AbstractZur Einbeziehung möglicher Effekte von organischer Substanz im Feststoff und im Eluat hinsichtlich der potentiellen Mobilisierung von hydrophoben organischen Schadstoffen aus Böden und Abfällen wird die Schadstoffmobilisierung durch tensidische Lösungen einerseits und jene durch naturnahe Lösungsvermittler andererseits verglichen. Während unter den Bedingungen des Elutionsverfahrens nach DIN 38414 Teil 4 gegenüber Eluenten mit organischen Naturstoffen erhebliche und in ihrem Ausmaß unterschiedliche Minderbefunde ermittelt werden, können deren mobilisierende Eigenschaften durch die eingesetzte tensidische Lösung gut simuliert werden.

Serum lipids during parenteral nutrition with a 10% lipid emulsion with reduced phopholipid emulsifier content in premature infants.

Fourteen premature infants (range 26 + 0 to 32 + 3), all but two appropriate for gestational age with a mean body weight of 1196 g (range 860 to 2770 g) received a 10% lipid emulsion. This lipid emulsion contained half of the formerly used phospholipid emulsifier concentration reducing the phospholipid/triglyceride ratio to the ratio used for the 20% lipid emulsion (0.06 instead of 0.12). Lipid emulsion was given over a 10 day period commencing at the third day of life with 0.5 g/kg/24 h which was increased daily up to a dose of 2.0-2.5 g/kg/24 h which was reached in all patients at the seventh day of the observation period. During this time mean serum concentrations of cholesterol increased non-significantly from 76.1 mg/dl (SD 33.7) before lipid emulsion to 86.1 mg/dl (SD 36.4) on day seven of the observation period. 13 of the 14 patients (97%) showed no pathological increase of their serum triglyceride concentration during lipid infusion. Mean serum triglyceride concentration increased from 65.3 mg/dl (SD 32.0 mg/dl) before the start of lipid emulsion to 102.6 mg/dl (SD 76.5) on day four (p < 0.05) but with no further significant increase. Lipid emulsions with 10% triglyceride but lower phospholipid content are tolerated without pathological increase in triglyceride or cholesterol serum concentration in the vast majority of premature newborns.

Alterations of erythrocyte morphology and lipid composition by hyperbilirubinemia

Morphology and membrane lipid composition of erythrocytes from neonates (jaundiced and healthy) and adults (before and after incubation with bilirubin) were studied. The morphological index, expressing the relative proportions of the different stages of cell distortion, and the membrane cholesterol, phospholipids and cholesterol/phospholipids molar ratio, were determined. In jaundiced neonates a significant increase in the morphological index ( P < 0.01) was found. After incubation with bilirubin, adult erythrocytes also showed an increase in the morphological index ( P < 0.01). Hemolysis occurred under these conditions, and the red cell ghosts obtained (vesicles) showed a rounded morphology. Higher cholesterol/phospholipid ratio and lower phospholipid content were found in jaundiced neonates compared with healthy babies ( P < 0.05) and adults ( P < 0.01), as well as in the cells ( P < 0.05) and vesicles ( P < 0.01) obtained after bilirubin incubation. Bilirubin cytotoxicity may occur in a stepwise manne: deposition of bilirubin in membrane produces echinocytosis, which is followed by disintegration of the lipid bilayer with loss of phospholipids from the membrane.

Molecular mechanisms of activated protein C resistance. Properties of factor V isolated from an individual with homozygosity for the Arg506 to Gln mutation in the factor V gene.

Resistance to activated protein C (APC), which is the most prevalent pathogenetic risk factor of thrombosis, is linked to a single point-mutation in the factor V (FV) gene, which predicts replacement of Arg (R) at position 506 with a Gln (Q). This mutation modifies one of three APC-cleavage sites in the heavy chain of activated FV (FVa), suggesting that mutated FVa (FVa:Q506) is at least partially resistant to APC-mediated degradation. To elucidate the molecular mechanisms of APC-resistance and to investigate the functional properties of FV in APC resistance, FV:Q506 was purified from an individual with homozygosity for the Arg to Gln mutation. Intact and activated FV:Q506 were demonstrated to convey APC resistance to FV-deficient plasma. Thrombin- or factor Xa-activated FV:Q506 were found to be approx. 10-fold less sensitive to APC-mediated degradation than normal FVa, at both high and low phospholipid concentrations. The degradation pattern observed on Western blotting suggested that FVa:Q506 was not cleaved at position 506. However, it was slowly cleaved at Arg306, which explains the partial APC sensitivity of FVa:Q506. FV is initially activated during clotting and then rapidly inactivated in a process which depends on the integrity of the protein C anticoagulant system. During clotting of APC-resistant plasma, FV:Q506 was activated in a normal fashion, but then only partially inactivated. In conclusion, the reduced sensitivity of FVa:Q506 to APC-mediated degradation is the molecular basis for the life-long hypercoagulable state which constitutes a risk factor for thrombosis in APC-resistant individuals.

Impairment of galactolipid biosynthesis in tomato pericarp at chilling temperature

Pericarp lipids of mature-green tomato fruits of three cultivars of Lycopersicon esculentum Mill., and one of L. esculentum × pimpinellifolium were analyzed to clarify the relationship between membrane lipid composition and chilling sensitivity. Only the hybrid cultivar, <New York 280> (<NY>), ripened normally at 20 °C after 16 d of chilling at 4 °C. At low temperature phospholipid content was maintained, while galactolipid content markedly declined in the four cultivars. Phosphatidylcholine content and the 18:3 content of phosphatidylcholine decreased slightly in <NY> while they increased slightly in sensitive <Early Cherry> (<EC>) during chilling. Before chilling, the molar ratio of di- to monogalactosyldiacylglycerol of chilling-tolerant <NY> was double that of the sensitive cultivars. This ratio increased only in <NY> during chilling. Diacylglycerol content decreased at 4 °C, while free fatty acid content and the % of 16:0, 18:0 and 18:1 in the free fatty acid fraction increased. The low degree of unsaturation of diacylglycerol and free fatty acids suggests that they were not the products of galactolipid catabolism during chilling. Phosphatidyl-glycerol fatty acid composition was similar in the four cultivars under all treatments. Taken together, our data indicate that phosphatidylglycerol is not a factor differentiating fruit-chilling sensitivity in tomato cultivars. Galactolipid biosynthesis may be impaired at chilling temperature; sensitive steps may be the acylation of glycerol-3-phosphate and the transfer of phosphatidylcholine diglyceride moieties to monogalactosyldiacylglycerol.