Cutaneous mastocytosis: A dermatological perspective.

Mastocytosis is a rare disease characterised by expansion and collection of clonal mast cells in various organs including the skin, bone marrow, spleen, lymph nodes and gastrointestinal tract. The prevalence of mastocytosis has been estimated to be one in 10 000, while the estimated incidence is one per 100 000 people per year. Cutaneous mastocytosis is classified into (i) maculopapular cutaneous mastocytosis, also known as urticaria pigmentosa; (ii) diffuse cutaneous mastocytosis; and (iii) mastocytoma of the skin. In adults, cutaneous lesions are usually associated with indolent systemic mastocytosis and have a chronic evolution. Paediatric patients, on the contrary, have often cutaneous manifestations without systemic involvement and usually experience a spontaneous regression. Diagnosis of cutaneous mastocytosis may be challenging due to the rarity of the disease and the overlap of cutaneous manifestations. This short review describes pathogenesis and clinical aspects of cutaneous mastocytosis with a focus on diagnosis and currently available therapies.

Endothelial lipase activity predicts high-density lipoprotein catabolism in hemodialysis: novel phospholipase assay in postheparin human plasma.

OBJECTIVE: A novel phospholipase assay was used to measure for the first time the behavior of endothelial and hepatic phospholipase activities in postheparin human plasma of hemodialyzed patients and its relationship with atherogenic and antiatherogenic lipoprotein levels. METHODS AND RESULTS: Endothelial and hepatic phospholipase activity was assessed in a total SN1-specific phospholipase assay, using (1-decanoylthio-1-deoxy-2-decanoyl-sn-glycero-3-phosphoryl) ethylene glycol as the substrate. Hemodialyzed patients presented lower values of total and hepatic phospholipase activity than controls: 4.4 (1.9-9.0) versus 7.5 (3.6-18.0) and 2.6 (0.7-6.2) versus 6.6 (1.3-15.2) µmol of fatty acid released per milliliter of postheparin plasma per hour, respectively (P<0.001); however, endothelial lipase (EL) phospholipase activity was increased in patients: 1.7 (0.8-3.0) versus 1.1 (0.1-2.7) µmol of fatty acid released per milliliter of postheparin plasma per hour (P=0.008). EL was negatively associated with high-density lipoprotein (HDL)-cholesterol (r=-0.427; P=0.001), and apolipoprotein A-I levels, total phospholipase, and hepatic lipase activity were directly associated with low-density lipoprotein-cholesterol and apolipoprotein B. The association of EL and HDL-cholesterol remained significant when adjusting for waist circumference (ß=-0.26; P=0.05), and the effect of hepatic lipase on low-density lipoprotein-cholesterol continued after adjusting for age (ß=0.46; P= 0.001). CONCLUSIONS: Our results support the hypothesis that EL is the predominant enzyme responsible for lipolytic catabolism of HDLs in hemodialyzed patients and resolve the apparent paradox observed between low hepatic lipase activity and decreased HDL-cholesterol levels observed in these patients. In addition, the ability to assess total hepatic lipase and EL phospholipase activity in plasma will increase our knowledge of the mechanisms involved in controlling HDL levels and cardiovascular risk in hemodialyzed patients, as well as other populations with low levels of HDL-cholesterol.

Involvement of a phospholipase D in the mechanism of action of granulocyte-macrophage colony-stimulating factor (GM-CSF): priming of human neutrophils in vitro with GM-CSF is associated with accumulation of phosphatidic acid and diradylglycerol.

The generation of diradylglycerol (DRG) and phosphatidic acid (PdtOH) was investigated in neutrophils primed with granulocyte-macrophage colony-stimulating factor (GM-CSF). Mass accumulation of DRG and PdtOH was measured using reversed-phase high performance liquid chromatography and thin layer chromatography, respectively. GM-CSF had no direct effect on the levels of PdtOH and DRG, but it increased PdtOH generation and the late phase of DRG accumulation in human neutrophils stimulated with FMLP. The elevation of the mass of PdtOH peaked approximately 100 s and clearly preceded that of DRG, which peaked at 150 s. The diacylglycerol kinase inhibitor R59022 enhanced the sustained increase in DRG but did not produce a parallel inhibition in PdtOH production. GM-CSF was without effect on the level of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and did not affect the liberation of Ins(1,4,5)P3 induced by FMLP. These findings exclude the involvement of the PtdIns(4,5)P2-specific phospholipase C/diacylglycerol pathway in the sustained phase of DRG accumulation. The early (30-s) appearance of PdtOH clearly suggests that GM-CSF enhanced FMLP receptor-linked phospholipase D (PLD) generation of PdtOH. PLD was assessed more directly by formation of labeled phosphatidylethanol (PEt) through PLD capacity of catalyzing a trans-phosphatidylation in presence of ethanol. The formation of PEt associated with a concomitant decrease in PdtOH directly demonstrated that the mechanism by which GM-CSF enhances PdtOH production is activation of a PLD active on phosphatidylcholine. This study provides evidence that the mechanism of action of GM-CSF involves upregulation of PLD activity leading to enhanced generation of PdtOH and DRG in FMLP-stimulated neutrophils. These findings may provide the basis for several of the priming effects of GM-CSF.

Metabolically unhealthy overweight individuals have high lysophosphatide levels, phospholipase activity, and oxidative stress.

BACKGROUND & AIMS: Metabolically unhealthy overweight (MUO) individuals and metabolically healthy overweight (MHO) individuals differ in biomarkers of atherogenesis. Metabolomic approaches enable studies of the metabolic variables underlying these differences. METHODS: We determined the metabolomes in plasma samples from 34 MUO and 34 MHO individuals matched for sex, age, and body mass index (BMI) to identify potential metabolic markers or pathways associated with atherogenic traits. RESULTS: This analysis revealed that the MUO group had significantly higher levels of glycolic acid, 6 lysophosphatidylethanolamines (lysoPEs), and 12 lysophosphatidylcholines (lysoPCs). Although the two groups had similar total body fat percentages and lean body masses, MUO individuals had larger visceral fat areas (VFAs). They also had greater circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and higher levels of oxidized low-density lipoprotein (ox-LDL) and urinary 8-epi-prostaglandin F2α (8-epi-PGF2α), reflecting higher risks for oxidative and lipid-related tissue damage. The following measures were positively correlated: VFA and ox-LDL; ox-LDL and Lp-PLA2 activity; and lysoPC, lysoPE, and 8-epi-PGF2α levels. Chronic plasma lysoPC increases were associated with atherogenic traits, higher levels of mean ox-LDL, 8-epi-PGF2α, Lp-PLA2, and visceral fat accumulation in MUO compared to MHO individuals. CONCLUSIONS: This panel of plasma metabolites distinguishes MUO from MHO individuals and will enable future research on fat dysregulation and obesity.

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils.

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.

A glycan-phosphatidylinositol-specific phospholipase D in human serum.

A group of proteins anchored to the cell by phosphatidylinositol (PI) has recently been identified. The significance of this new class of membrane anchor is unknown; one possibility is that it facilitates release of the molecule by phospholipases. In fact, phospholipase C enzymes specific for the complex carboxyl-terminal glycolipids of these proteins have been isolated from African trypanosomes and from hepatocyte plasma membranes. This study reports the discovery of a glycan-PI-specific phospholipase D in human serum that cleaves both the membrane form of the variant surface glycoprotein of African trypanosomes and its glycolipid precursor, but not phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol. Decay-accelerating factor, another PI-anchored molecule, is also cleaved by the enzyme and converted from a hydrophobic to a soluble protein. The enzyme is Ca2+-dependent, heat labile, and not affected by the inhibitor of serine proteases, phenylmethylsulfonylfluoride. Its function is not known, but the present findings indicate that it participates in the metabolism of glycolipid-anchored membrane proteins.

Isolation of human eosinophil phospholipase D.

Phospholipase D preferentially contained in human eosinophil polymorphonuclear leukocytes as compared to other leukocytes was isolated by sequential asion and cation exchange chromatography and gel filtration. The purified eosinophil enzyme specifically liberated choline from I-alpha-phosphatidyl choline with a pH optimum of 4.5-6.0 and exhibited a pI of 5.8-6.2 on polyacrylamide-gel isoelectric focusing, which are properties shared by phospholipase D from plant sources; however, its apparent mol wt of 60,000 is approximately one-half that of the plant enzymes. Eosinophil and cabbage phospholipase D inactivated a partially purified rat platelet-activating factor (PAF) in a time- and dose-dependent reaction. The cleavage of this PAF activity was attributed to the inherent phospholipase D activity of the eosinophil enzyme since the two activities chromatographed together at each purification step, and there was apparent reciprocal inhibition of choline-generating activity by PAF and of PAF-inactivating activity by phosphatidyl choline. Thus, possible regulatory functions of the eosinophil in immediate hypersensitivity reactions include inactivation of a PAF by phospholipase D as well as degradation of slow-reacting substance of anaphylaxis by arylsulfatase B.

Phospholipid metabolism in stimulated human platelets. Changes in phosphatidylinositol, phosphatidic acid, and lysophospholipids.

Endogenous phospholipid metabolism in stimulated human platelets was studied by phosphorus assay of major and minor components following separation by two-dimensional thin-layer chromatography. This procedure obviated the use of radioactive labels. Extensive changes were found in quantities of phosphatidylinositol (PI) and phosphatidic acid (PA) as a consequence of thrombin or collagen stimulation. Thrombin addition was followed by rapid alterations in the amount of endogenous PI and PA. The decrease in PI was not precisely reciprocated by an increase in PA when thrombin was the stimulus. This apparent discrepancy could be explained by removal of a transient intermediate in PI metabolism, such as diglyceride, formed by PI-specific phospholipase C (Rittenhouse-Simmons, S., J. Clin. Invest.63: 580-587, 1979). Diglyceride would be unavailable for PA formation by diglyceride kinase, if hydrolyzed by diglyceride lipase (Bell, R. L., D. A. Kennerly, N. Stanford, and P. W. Majerus. Proc. Natl. Acad. Sci. U. S. A.76: 3238-3241, 1979) to yield arachidonate for prostaglandin endoperoxide formation. Thrombin-treated platelets also accumulated lysophospho-glycerides. Specifically, lysophosphatidyl ethanolamines accumulated within 15s following thrombin addition. Fatty acid and aldehyde analysis indicated phospholipase A(2) activity, with an apparent preference for diacyl ethanolamine phosphoglycerides. In the case of collagen, these changes occurred concomitantly with aggregation and consumption of oxygen for prostaglandin endoperoxide formation.THESE STUDIES OF ENDOGENOUS PHOSPHOLIPID METABOLISM PROVIDE INFORMATION SUPPORTING THE EXISTENCE OF TWO PREVIOUSLY POSTULATED PATHWAYS FOR LIBERATION OF ARACHIDONIC ACID FROM PLATELET PHOSPHOLIPIDS: (a) the combined action of PI-specific phospholipase C plus diglyceride lipase yielding arachidonate derived from PI; and (b) a phospholipase A(2) acting primarily on diacyl ethanolamine phosphoglyceride.

Characterization of serum platelet-activating factor (PAF) acetylhydrolase. Correlation between deficiency of serum PAF acetylhydrolase and respiratory symptoms in asthmatic children.

Platelet-activating factor (PAF) acetylhydrolase has been recognized as an enzyme that inactivates PAF. We developed a convenient and reproducible method for determining human serum PAF acetylhydrolase activity. The assay was based on measurement of [14C]acetate produced from 1-O-alkyl-2-[14C]-acetyl-sn-glycero-3-phosphocholine upon precipitation of the complex of radioactive substrate and albumin with TCA. The apparent Km value of PAF acetylhydrolase (near the physiological concentration of serum protein) was 1.5 X 10(-4) M PAF. 32 subjects with serum PAF acetylhydrolase deficiency were found among 816 healthy Japanese adults. The low PAF acetylhydrolase activity in the deficient serum might not be due to the presence of enzyme inhibitor. Both the sensitivity to PAF and the metabolism of PAF in platelets from PAF acetylhydrolase-deficient subjects were almost the same as those of normal subjects. Deficiency in serum PAF acetylhydrolase appeared to be transmitted by autosomal recessive heredity among five Japanese families. Among healthy adults, healthy children, and asthmatic children, who were grouped into five classes on the basis of respiratory symptoms (remission, wheezy, mild, moderate, and severe groups), the probability of PAF acetylhydrolase deficiency was significantly higher in groups with severe symptoms (moderate and severe) (P less than 0.01). These results suggest that deficiency of serum PAF acetylhydrolase might be one of the factors leading to severe respiratory symptoms in asthmatic children.

Hepatic lipase gene therapy in hepatic lipase-deficient mice. Adenovirus-mediated replacement of a lipolytic enzyme to the vascular endothelium.

Hepatic lipase (HL) is an endothelial-bound lipolytic enzyme which functions as a phospholipase as well as a triacylglycerol hydrolase and is necessary for the metabolism of IDL and HDL. To evaluate the feasibility of replacing an enzyme whose in vivo physiologic function depends on its localization on the vascular endothelium, we have infused recombinant replication-deficient adenovirus vectors expressing either human HL (HL-rAdV; n = 7) or luciferase cDNA (Lucif-rAdV; n = 4) into HL-deficient mice with pretreatment plasma cholesterol, phospholipid, and HDL cholesterol values of 176 +/- 9, 314 +/- 12, and 129 +/- 9, respectively. After infusion of HL-rAdV, HL could be detected in the postheparin plasma of HL-deficient mice by immunoblotting and postheparin plasma HL activities were 25,700 +/- 4,810 and 1,510 +/- 688 nmol/min/ml on days 5 and 15, respectively. Unlike the mouse HL, 97% of the newly synthesized human HL was heparin releasable, indicating that the human enzyme was virtually totally bound to the mouse vascular endothelium. Infusion of HL-rAdV in HL-deficient mice was associated with a 50-80% decrease in total cholesterol, triglyceride, phospholipids, cholesteryl ester, and HDL cholesterol (P < 0.001) as well as normalization of the plasma fast protein liquid chromatography lipoprotein profile by day 8. These studies demonstrate successful expression and delivery of a lipolytic enzyme to the vascular endothelium for ultimate correction of the HL gene defect in HL-deficient mice and indicate that recombinant adenovirus vectors may be useful in the replacement of endothelial-bound lipolytic enzymes in human lipolytic deficiency states.

Intestinal phospholipase, a novel enzyme.

We evaluated phospholipase activity in the intestine of rats and other species. Phospholipase activity was assayed by a surface barostat technique or an egg yolk titration system. Mucosal activity was found only by the surface barostat technique with phosphatidylglycerol as substrate; it was not found with phosphatidylcholine as substrate in assays by either technique. In gut luminal fluid activity was found when both phosphatidylcholine and phosphatidylglycerol were used as substrate in assays by the surface barostat technique, and phosphatidylcholine as substrate yielded activity in egg yolk titration. In rats in which pancreatic juice had been diverted, mucosal and gut luminal phospholipase activity was greater than in controls, thus demonstrating that enzyme activity was not due to pancreatic phospholipase. Bacterial origin of phospholipase activity was excluded in that phospholipase activity was found in germ-free rats; gastric and salivary gland origins were excluded in that continued phospholipase activity was found in rats with gastric fistula. The physiological importance of the enzyme was established by the finding that rats with pancreatic fistula absorbed 111 mumol of phosphatidylcholine and that controls absorbed 119 mumol of a 135-mumol load. Activity was found to be three times greater in the distal than in the proximal intestine; in cryptal cells it was 10 times greater than in villus tip cells. 65% of the activity in the gut lumen was tightly bound to particulate matter. We propose that intestinal phospholipase may be important in gut bacterial control, in the digestion of vegetable matter (phosphatidylglycerol is a major phospholipid in both plants and bacteria), and in the digestion of phospholipids in the gut lumen.

Abnormal lipoprotein lipase in familial exogenous hypertriglyceridemia.

A 5-yr old male proband and his sister have had hypertriglyceridemia and hepatosplenomegaly since birth. When studied on a metabolic ward, they demonstrated rapid decreases in serum triglycerides on 3 g fat/day diets. Oral glucose tolerance tests were normal. Postheparin lipolytic activity (PHLA) against chylomicrons was virtually absent in both children whereas the mother and a normolipemic sister had levels approximately 50% normal. However, all four had a normal PHLA against commercial triglyceride emulsion (Intralipid). Two unrelated children from different kindreds of typical type 1 hyperlipoproteinemia and two patients with acquired type V hyperlipoproteinemia had deficient PHLA against both substrates. No inhibitors of PHLA could be demonstrated in the proband's plasma, and his own PHLA could not be enhanced by either normal concentrated plasma or pooled d > 1.063 lipoprotein fraction. The proband's postheparin plasma required almost 20 times the normal chylomicron-triglyceride concentration to reach one-half maximal lipase velocity. Both affected siblings showed heavy pre-beta lipoprotein electrophoretic bands plus chylomicrons in their fasting plasmas while ingesting a 33% carbohydrate, 30% fat diet. Incubation of their postheparin plasma with S(f) > 400 chylomicrons in vitro produced a smaller S(f) 20-400 "remnant" with pre-beta electrophoretic mobility that was not seen under the same conditions when normal postheparin plasma was used. Postheparin monoglyceridase and phospholipase activities were either normal or only moderately decreased when determined with appropriate artificial substrates. These data are consistent with either (a) a mutant gene producing a lipoprotein lipase with unusual substrate specificities or (b) an absolute deficiency of normal lipoprotein lipase with a compensatory increase in some other postheparin triglyceridase.

Selective release of archidonic acid from the phospholipids of human platelets in response to thrombin.

Arachidonic acid is unique amongst human platelet fatty acids in that it is the precursor of prostaglandins and thromboxanes. Since a number of these oxygenated products of arachidonic acid have potent effects on platelet function, an understanding of the metabolsim of their precursor is important. Human platelets have a mechanism for incorporating arachidonic acid from plasma into their phospholipids and, in response to thrombin, they reveal mechanisms for hydrolyzing this arachidonic acid from platelet phosphatidylcholine and phosphatidylinositol. This report deals with the specificity of these mechanisms. The present studies show that human platelets contain phospholipase A2 activities that preferentially release arachidonic acid. One of these activities specifically utilizes 1-acyl-2-arachidonyl-phosphatidyl-choline. Another utilizes platelet phosphatidylinositol and/or phosphatidylserine, both of which are highly enriched with arachidonic acid.

[Phospholipase A2 activity in rat serum. Association of two proteins (author's transl)]. / Activité phospholipasique A2 du sérum de rat. Association de deux protéines.

In the rat, blood coagulation is accompanied by activation of a plasma phospholipase A2 due to a factor contained in blood platelets and released in plasma by platelet lysis. This activity can be obtained in vitro by treating plasma devoid of platelets with a platelet lysate. The results reported in this communication show the activity of serum phospholipase to result from an association of two proteins, inactive when isolated, one originating from plasma, the other from platelets, which can be broken down by fractionation on a column of Blue Sepharose CL-6B.

Modulation of purified phospholipase A2 activity from human platelets by calcium and indomethacin.

A membrane bound phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human platelets has been purified 3500-fold, and partially characterized. Phospholipase A2 activity was assayed using [1(-14)C] oleate-labeled Escherichia coli or sonicated dispersions of synthetic phospholipids. The 2-acyl specificity of the phospholipase activity was confirmed using phosphatidylethanolamine labeled in the C-1 position as substrate. The purified enzyme was maximally active between pH 8.0 and 10.5, and had an absolute requirement for low concentrations of Ca2+. Indomethacin, but not aspirin, inhibited phospholipase A2 activity.

Resolution into two different forms and study of the properties of phosphatidylinositol-specific phospholipase C from human platelet cytosol.

Two forms of phosphatidylinositol-specific phospholipase C from human platelet cytosol were resolved by DEAE-cellulose chromatography and purified further by hydrophobic chromatography. Both forms utilized phosphatidylinositol as the best substrate. However, the enzyme did not distinguish 2-arachidonylphosphatidylinositol from 2-oleoylphosphatidylinositol although the former substrate was known to be a predominant species in human platelets. Both forms exhibited pH optimum at 7.0. Both activities were inhibited completely by 1 mM EDTA and the inhibited preparations could be restored to full activity or to 60% by free Ca2+ or Co2+, respectively, at 100 microM. Higher concentrations of either ion were inhibitory. Other metal ions were ineffective. Addition of calmodulin in the presence of Ca2+ did not show any additional effect. Both forms were inhibited comparably by various phospholipids, fatty acids and detergents, suggesting that phosphatidylinositol in membranes might be a poor substrate for the enzyme. Initiation of phosphatidylinositol breakdown through the phospholipase C pathway may require additional activator(s). A variety of anti-platelet drugs, including phenylthiazines, local anesthetics and mepacrine, were found to be potent inhibitors of the enzyme, suggesting that these drugs might inhibit platelet function by inhibiting the early phase of arachidonate release.

Effect of different physical states of phospholipid substrates on partially purified platelet phospholipase A2 activity.

Partial purification of alkaline phospholipase A2 (EC 3.1.1.4) from rabbit platelets was carried out and the effect of different physical states of the substrate phosphatidylcholine on the activity was investigated. (1) The enzyme was purified about 1020-fold by means of Sephadex gel chromatography after extraction from a particulate fraction of rabbit platelets, followed by CM-cellulose chromatography, and had a molecular weight of approx. 12 000 as determined by gel chromatography. (2) The activity of the purified enzyme was enhanced by the addition of detergents. Sodium deoxycholate and sodium cholate markedly stimulated the activity, and the effect of these substances was observed well below the critical micelle concentrations. Triton X-100 stimulated the activity moderately, and the activation was observed only above the critical micelle concentration. (3) The addition of negatively charged phospholipids to the substrate egg phosphatidylcholine induced a moderate activation of hydrolysis. (4) The addition of long-chain cation to the substrate induced an inhibition of the activity, whereas the addition of long-chain anion activated the hydrolysis of egg phosphatidylcholine, but did not activate the hydrolysis of phosphatidylcholine in the total lipid extract of rabbit platelets. (5) Hydrolysis of dimyristoyl phosphatidylcholine increased in the temperature region of the phase transition of the substrate. Addition of cholesterol at the concentration of 20 mol% diminished the effect of phase transition. (6) Release of [1-14C]arachidonic acid from an equimolar mixture of egg phosphatidylcholine with dipalmitoyl or distearoyl phosphatidylcholine was activated at the temperature of 0 degrees C or 20 degrees C, respectively. From these results, we suggest that platelet phospholipase A2 can be activated to release fatty acids from the platelet phospholipids at the domains within membranes, where exist the structural irregularities and/or accumulation of negative charge within the bilayers.

Evidence for active phospholipase A in mouse plasma.

Mouse, rat and human plasma were exposed to minimum concentrations of disulphide or minimum pre-incubation at 55 degrees C in order to inhibit lecithin : cholesterol acyltransferase activity completely. The plasma samples were subsequently incubated at 37 degrees C and changes in individual phospholipid concentrations determined. Significant utilization of phosphatidylcholine and formation of lysophosphatidylcholine occurred only in disulphide-treated mouse plasma and this was accompanied by a decrease in total phospholipid concentration. When disulphide-treated mouse plasma was incubated with [U-14C]phosphatidylcholine radioactivity was additionally recovered in the lysophosphatidylcholine, non-esterified fatty acid and glycero-3-phosphocholine fractions; maximum conversion occurred at close to physiological pH. These observations suggest that phospholipase A and lysophosphatidylcholine hydrolase enzymes are active in mouse plasma but that phospholipase A is either absent or inactive in rat and human plasma.

Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma.

A vasoactive lipid present in rat plasma that had been incubated at 37 degrees C for 48 h was identified as lysophosphatidic acid with the following fatty acid composition: palmitic acid (25.2%), stearic acid (8.4%), oleic acid (7.0%), linoleic acid (44.4%) and arachidonic acid (11.9%). The involvement of lysophospholipase D in the production of lysophosphatidic acid was suggested on the basis of extensive analyses of phospholipids in the plasma. Results indicated that lysophospholipase D hydrolyzed polyunsaturated lysophosphatidylcholines preferentially to the saturated species. Exogenously added platelet activating factor was found to be first converted to its lyso derivative by an acetylhydrolase in rat plasma and then degraded by lysophospholipase D to 1-alkyllysophosphatidic acid to a similar extent with exogenously added lyso derivative of platelet activating factor; their percent conversions to lysophosphatidic acid were higher than those of saturated 1-acyllysophosphatidylcholines, but lower than those of polyunsaturated 1-acyllysophosphatidylcholines.

Endogenous suppression of neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes.

Phospholipase A2 activity was measured in homogenized and acid-extracted human polymorphonuclear leukocytes using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate. In whole homogenate and in the supernatant and particular fractions separated by centrifugation at 150,000 X g, phospholipase activity was barely detectable (1-4 pmol/h per 10(6) cell equivalents). By contrast, acid extracts of these fractions contained over 10-times as much phospholipase activity in the dialyzed supernatants (20-300 pmol/h per 10(6) cell equivalents), whereas phospholipase inhibitor(s) were found in the sediment. The acid-solubilized phospholipase A2 activity was absolutely Ca2+-dependent and optimal at pH 7.0-7.5 with 1.0 mM added Ca2+. Addition of the resuspended sediment of the acid extract dose-dependently suppressed phospholipase activity in the supernatant; less than equivalent amounts were sufficient to inhibit 95%. Suppressor activity was lipid-extractable. After thin layer chromatography of lipid extracts, the bulk of inhibitory activity was recovered from the free fatty acid region. Analysis of the fatty acids by gas liquid chromatography showed that 63% were unsaturated. All unsaturated fatty acids tested were potent inhibitors of phospholipase A2 activity (IC50 3-10 microM). Oleoyl-CoA, hydroxyeicosatetraenoic acids and leukotriene D4 were also inhibitory, while methyl oleate, saturated fatty acids and the prostaglandins E2 and F2 alpha had no effect. These in vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.