Platelet-activating factor type activity in plasma from patients with septicemia and other diseases

The purpose of the present study was to determine whether increased levels of platelet-activating factor (PAF) type activity can be detected in plasma from patients with septicemia and other diseases. A level of PAF below 0.5 ng/mL of plasma was considered normal. We found that plasma from a patient with adverse anaphylactoidic reaction to intravenous analgetics contained 2.1 ng PAF/mL. In seven patients with septicemia, including urosepsis, endocarditis and peritonitis, and with positive blood culture, increased plasma PAF levels (1–20 ng PAF/mL) were observed. Other patients with clinical indications of septicemia had negative blood cultures and/or increased levels of C-reactive protein (CRP). Yet, in the plasma from these patients, no increased PAF levels were detected under the assay conditions used. Two patients with allergic asthma, requiring treatment with steroids, had no measurable plasma PAF. In the plasma from a patient with idiopathic thrombocytopenic purpura (ITP) only an “endogenous” inhibitor of PAF induced platelet aggregation was initially observed. In spite of this, the patient responded to treatment with the PAF antagonist WEB 2086 with a dramatic increase in platelet count (Lohmannet al., Lancet ii, 1147, 1988). Thereafter, also increased PAF levels (3.3 ng PAF/mL) were detected in plasma, although some “endogenous” inhibitor of PAF was still present. In conclusion, increased PAF levels in plasma from patients support a role of PAF in certain human disease states, such as in anaphylactoid reaction, sepsis and septic shock. The type, relevance and specificity of endogenous inhibitors of PAF deserve further study. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

A simple and precise method for the routine determination of platelet-activating factor in blood and urine

A simple and precise method is described for the measurement of platelet-activating factor (PAF) in blood and urine. The method involves the isolation of PAF from blood samples by two successive steps. In the first step, blood proteins are precipitated with ethanol and the “free” PAF, i.e. the PAF which is extractable with ethanol, is recovered. In the second step, “bound” PAF, i.e., PAF not extractable with ethanol, is extracted from the protein precipitate with chloroform/methanol/water. The extraction of PAF from urine samples requires only the ethanol extraction step. “Free” and “bound” PAF are then each fractionated by silicic acid column chromatography, and the methanol/water eluent containing PAF is then further fractionated by high-performance liquid chromatography using an isocratic solvent system of acetonitrile/methanol/water. PAF is then quantitated by measuring its ability to induce platelet aggregation in an aggregometer. Application of the method to blood and urine samples from twenty-three healthy volunteers revealed PAF levels in blood of 140–480 pg/mL (630–254.4 pg “free” PAF/mL and 64–225.6 pg “bound” PAF/mL), and of 1.2–4.0 pg PAF/mL in urine. The method overcomes various technical problems and was shown to be very precise. It should prove useful for monitoring PAF levels in various disease conditions.     

The effect of short-term diets rich in fish, red meat, or white meat on thromboxane and prostacyclin synthesis in humans

Foods which increase tissue arachidonic acid levels have been proposed to increase thrombosis tendency, presumably through increased platelet aggregation. This study examined the effect of doubling the dietary arachidonic acid (20∶4n−6) using meat- or fish-based diets on the systemic production of prostacyclin (PGl₂) and thromboxane (TXA₂) in 29 healthy, nonsmoking adults. There were three, 3-wk low-fat dietary periods (<15% energy as fat) in which subjects consumed a vegetarian diet for 1 wk followed by 2 wk on diets containing meat or fish as sources of 20∶4n−6. Between each diet period, there was a 3-wk washout period, during which subjects returned to their normal diets. The level of 20∶4n−6 consumed during the last 2 wk of each study was approximately double the usual intake (mean 140 mg/d), while the mean eicosapentaenoic acid (20∶5n−3) content of the diets varied from 1 mg/d on the white meat diet to 70 mg/d on the red meat diet and to 847 mg/d on the fish diet. The serum phospholipid (PL) 20∶4n−6/20∶5n−3 ratios were 11∶1 on the vegetarian diet, 15∶1 on the white meat diet, 8∶1 on the red meat diet, and 2∶1 on the fish diet (P<0.001). Neither white nor red meat diets affected platelet 20∶4n−6 levels, platelet aggregation, ex vivo platelet TXB₂ production, or the systemic PGl₂ or TXA₂ production as measured by gas chromatography-mass spectrometry analysis of the excretion levels of the principal urinary metabolites 2,3-dinor-6-keto-PGF_1α (PGl₂-M) and 11-dehydro-TXB₂ (TXA₂-M), respectively. The fish diet decreased the 20∶4n−6/20∶5n−3 ratio in platelet PL from the baseline level of 45∶1 to 13∶1 (P<0.001), had no effects on platelet aggregation, but significantly decreased platelet TXB₂, production (collagen-stimulated) and TXA₂-M production, while PGl₂-M levels were unaltered. These results indicate that short-term diets which double the usual 20∶4n−6 intake using white meat (175–330 g/d) or red meat (275–530 g/d) are not associated with an increased TXA₂ production, but this does not rule out the adverse effects of 20∶4n−6 at higher levels in the diet, or for more prolonged periods. Short-term diets containing fish (100–200 g/d with 90–210 mg/d 20∶4n−6 and approximately 650–1000 mg/d 20∶5n−3) led to significant increases in platelet 20∶5n−3 levels and a decrease in the ex vivo and systemic TXA₂ production.     

Inhibitor(s) of platelet-activating factor (PAF) in human saliva

Quantitation of platelet-activating factor (PAF) in human saliva samples by radioimmunoassay indicated there was, at times, sufficient PAF present to aggregate platelets. However, in certain samples, we observed little or no aggregation, and furthermore, these samples were found to inhibit aggregation induced by PAF (200 pg). Chromatographic fractionation of pooled saliva increased the PAF activity 4-fold, and the observed inhibitory activity was found to co-migrate with the fatty acids. The inhibitory fraction was found to be active against platelet aggregation induced by arachidonic acid (3.4 nmole) as well as PAF (25 pg), but not thrombin (20 mU). These results indicate the existence of a PAF inhibitor in saliva, which may explain why potentially toxic levels of PAF can occur in the saliva of normal, healthy individuals. These findings also highlight an important advantage of the radioimmunoassay over platelet aggregation for the quantitation of PAF in, at least, some biological fluids. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor (PAF) receptor antagonists inhibit arachidonic acid induced platelet aggregation in rabbit whole blood

The potency of several platelet-activating factor (PAF) receptor antagonists was measured by observing their inhibitory effects against PAF induced platelet aggregation. Their selectivity was assessed by monitoring their effect on platelet aggregation induced by arachidonic acid (AA) and adenosine diphosphate (ADP). The antagonists inhibited platelet aggregation induced at the PAF EC₅₀ (0.023 μM) with the following rank order of potency: WEB 2086> WEB 2170> SRI 64–412> SRI 63–675> BN 52021>kadsurenone> SRI 63–441> alprazolam. While the antagonists had no inhibitory effect at the EC₅₀ for ADP (10 μM), they did inhibit platelet aggregation induced at the EC₅₀ for AA (55 μM). However, there was considerable variability in the slope of the inhibitory response and the relative potency of each antagonist against PAF induced platelet aggregation as compared to AA induced platelet aggregation. The antagonist IC₅₀ (μM) against PAF and AA were as follows, with those that showed significantly different (p<0.01) slopes indicated by an asterisk: SRI 63-441^* (3.8, 15.1); SRI 63-675 (1.4, 36.2); SRI 64-412 (0.5, 10.5); BN 52021^* (2.4, 58.9); kadsurenone^* (2.8, 28.3); alprazolam^* (10, 25); WEB 2086 (0.055, 0.220), and WEB 2170 (0.107, 0.534). Therefore, in rabbit whole blood the antagonists were potent, although not completely selective, inhibitors of PAF induced platelet aggregation. These results suggest that the mode of action of PAF and AA induced platelet aggregation may share some common features. However, since the slope of the inhibitory response against PAF and AA for some antagonists differed, mechanistic differences in their action appear to exist. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Effects of dietary n−3 and n−6 polyunsaturated fatty acids on macrophage phospholipid classes and subclasses

This study examined the effects of n−3 and n−6 polyunsaturated fatty acid alimentation on murine peritoneal macrophage phospholipids. Mice were fed complete diets supplemented with either corn oil predominantly containing 18∶2n−6, borage oil containing 18∶2n−6 and 18∶3n−6, fish/corn oil mixture containing 18∶2n−6, 20∶5n−3 and 22∶6n−3, or fish/borage oil mixture containing 18∶2n−6, 18∶3n−6, 20∶5n−3 and 22∶6n−3. After two weeks, the fatty acid levels of glycerophosphoserines (GPS), glycerophosphoinositols (GPI), sphingomyelin (SPH), and of the glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) phospholipid subclasses were determined. We found that mouse peritoneal macrophage GPC contain primarily 1-0-alkyl-2-acyl (range for the dietary groups, 24.6–30.5 mol %) and 1,2-diacyl (63.2–67.2 mol %), and that GPE contains 1-O-alk-1-enyl-2-acyl (40.9–47.4 mol. %) and 1,2-diacyl (44.2–51.2 mol %) subclasses. In general, fish oil feeding increased macrophage 20∶5n−3, 22∶5n−3 and 22∶6n−3 levels while simultaneously reducing 20∶4n−6 in GPS, GPI, GPE and GPC subclasses except for 1-O-alk-1′-enyl-2-acyl GPC. Administration of 18∶3n−6 rich diets (borage and fish/borage mixture) resulted in the accumulation of 20∶3n−6 (2-carbon elongation product of 18∶3n−6) in most phospholipids. In general, the novel combination of dietary 18∶3n−6 and n−3 PUFA produced the highest 20∶3n−6/20∶4n−6 phospholipid fatty acid ratios. This study demonstrates that marked differences in the responses of macrophage phospholipid classes and subclasses exist following dietary manipulation. The reduction of 20∶4n−6, while simultaneously increasing 30∶3n−6 and n−3 PUFA levels, may be important in relation to the putative beneficial effects of 20∶3n−6 and fish oil on macrophage eicosanoid and platelet activating factor (PAF) biosynthesis.     

Chemical synthesis and physiological activity of sulfonium analogues of platelet activating factor

Phosphatidylsulfocholine (PSC), the sulfonium analogue of phosphatidylcholine (PC), occurs naturally in some diatoms. The replacement of the −N⁺(CH₃)₃ group by a −S⁺(CH₃)₂ results in an increase in the polar head group size in PSC relative to that of PC, consistent with the observed increase in permeability of PSC bilayers towards urea. It was of interest to see whether replacement of the −N⁺(CH₃)₃ group in platelet activating factor (PAF) by an −S⁺(CH₃)₂ group leads to any change in platelet aggregation or other physiological activity. Synthesis of the sulfonium analogue of PAF was carried out by suitable modifications of known procedures. The PAF-sulfonium analogue was found to have almost the same platelet aggregating activity as PAF itself, in the concentration range 1–20 μM, but a much lower activity in the range 0.01–1 μM. The analogue had little or no effect on the platelet aggregation activity of PAF when added in the concentration range 0.01–1 μM and had about half the hypotensive activity of PAF towards hypertensive CDF male rats. The sulfonium analogue, however, was much more cytotoxic to HL-60 cells than PAF itself, in the concentration range 0–15 μM; replacement of the acetate group by a benzyl group increased the cytotoxicity to the level of that of the methoxy analogue of PAF. Thus, replacement of the −N⁺(CH₃)₃ group by a −S⁺(CH₃)₂ group in the polar head group region of PAF results in a relatively small change in its platelet aggregation activity and a decrease in its hypotensive activity, but greatly increases its antitumor activity. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 8–12, 1989.     

Albumin-bound docosahexaenoic acid and collagen-induced human platelet reactivity

Anin vitro system designed to mimic the effect of various plasma nonesterified (polyunsaturated) fatty acids on platelet function and metabolism was employed. Human platelet aggregation induced by submaximal (1.8 μg/ml) collagen stimulation was significantly inhibited by 2 min preincubation with 20 μM albumin-bound docosahexaenoic acid (22∶6n−3) (DHA), but not by the other fatty acids tested. [³H]Phosphatidic acid (PA) formation, an indicator of phospholipase C activation following platelet stimulation, was moderately inhibited by eicosapentaenoic acid (20∶5n−3), 11,14,17-eicosatrienoic acid (20∶3n−3), dihomo-γ-linolenic acid (20∶3n−6), as well as DHA, but not by arachidonic acid (20∶4n−6); this inhibition of phospholipase C activation could not explain the differential effect of DHA on platelet aggregation. The decreased production of thromboxane A₂ (TxA₂), as assessed by [³H]12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) formation, may account for the inhibition of collagen-induced aggregation by 20 μM DHA. Surprisingly, preincubation with 40 μM albumin-bound DHA, even though resulting in greater inhibition of collagen-induced aggregation, had less impact on HHT formation. A small but significant increase in [³H]prostaglandin D₂ (PGD₂) levels following 3-min collagen stimulation may have contributed to the greater antiaggregatory effect of 40 μM DHA. It is concluded that increased plasma nonesterified DHA may contribute to the dampened platelet activation and altered metabolism following fish oil supplementation of the diet.     

Study on the microwave-assisted extraction of polyphenols from tea

This study demonstrated a promising method for quickly extracting tea polyphenol (TP) by microwave-assisted extraction (MAE) technology. Some influential parameters, including MAE temperature, microwave power, concentration of extraction solvent, MAE time and the solid/liquid ratio, were investigated. The optimum condition of MAE was obtained by dual extraction with 60% ethanol (v/v) and the solid/liquid ratio 1:12 g/mL at 80°C for 10 minutes under the microwave power 600 W. The yield of TP was 96.5% under the described condition. Compared with traditional methods, including hot reflux extraction (HRE), ultrasound-assisted extraction (UAE) and supercritical fluid extraction (SFE), the extraction time was saved 8 times than that of HRE, and the yield was increased by 17.5%. The extraction time at comparable levels of production was saved 2 times, and the energy consumption was one fourth that of UAE. The extraction time was saved 5 times than that of SFE, and the yield of TP was increased by 40%. Moreover, compared with MAE of TP studied by others, it decreased the solid/liquid ratio from 1 ∶ 20 to 1 ∶ 12 g/mL without 90-min pre-leaching time, and the yield of TP was increased by 6%–40%.     

Effect of the hetrazepinoic platelet-activating factor antagonist bepafant (WEB 2170) in models of active and passive anaphylaxis in mice and guinea pigs

The selective hetrazepinoic platelet-activating factor (PAF) antagonist WEB 2170 (Bepafant) was used to study the pathophysiological role of PAF in several models of anaphylaxis in mice and guinea pigs. In actively sensitized mice, the PAF antagonist WEB 2170 (1.0–10 mg/kgp.o.) protected mice from anaphylactic death in a dosedependent manner when the anaphylactic response was potentiated by the beta-receptor antagonist propranolol. When active anaphylaxis in guinea pigs was induced intravenously by 100 mg/kg ovalbumin (OA) in the presence of small doses of the antihistamine mepyramine, additional treatment with oral or intravenous WEB 2170 protected the guinea pigs from anaphylactic death. Also, the remaining anaphylactic bronchoconstriction and blood pressure changes (including anaphylactic hypotension) were attenuated. When guinea pigs were passively sensitized with a heterologous antibodyvia the tracheal route and then challenged by ovalbumin (100 mg/kgi.v.) 24 hr after sensitization in the presence of 0.003 mg/kgi.v. mepyramine, additional treatment with tracheal WEB 2170 at 0.1–1 mg/kg protected the guinea pigs dosedependently not only from anaphylactic death but also from a further decrease of respiratory flow and changes of blood pressure. Increased levels of PAF-like activity (20–50 ng PAF/whole lung) were detected in lungs removed from antigen-challenged animals. The results suggest a causative role for PAF in active and passive anaphylaxis. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Eskimo plasma constituents,dihomo-γ-linolenic acid,eicosapentaenoic acid and docosahexaenoic acid inhibit the release of atherogenic mitogens

Studies in man and laboratory animals suggest that ω3 polyunsaturated fatty acid consituents of fish oils have antiatherosclerotic properties. We have studied the effects of several such polyunsaturated fatty acids for ability to modify the in vitro release of mitogens from human platelets. Such mitogens may produce the fibroproliferative component of atherosclerotic plaques. Both 5,8,11,14,17-eicosapentaenoic acid (20∶5ω3) and 4,7,10,13,-16,19-docosahexaenoic acid (22∶6ω3), major constituents of fish oils, inhibited adenosine diphosphate-induced aggregation of platelets and the accompanying release of mitogens. These effects are dose dependent. Linolenic acid (18∶3ω3), the biosynthetic precursor of eicosapentaenoic acid, also inhibited platelet aggregation and mitogen release. Eicosapentaenoic acid also inhibited mitogen release from human monocyte-derived macrophages, which, in vivo, are an additional source of mitogens during atherogenesis. Potent inhibition of human platelet aggregation and mitogen release was also seen with dihomo-γ-linolenic acid (8,11,14-eicosatrienoic acid 20∶3ω6), whose levels are reportedly elevated in Eskimos subsisting on marine diets. We conclude that diets that elevate plasma and/or tissue levels of eicosapentaenoic acid, docosahexaenoic acid and dihomo-γ-linolenic acid precursor γ-linolenic acid (18∶3ω6) may exert antiatherosclerotic effects by inhibiting the release of mitogens from platelets and other cells.     

Dietary saturated,monounsaturated, n−6 and n−3 fatty acids,and cholesterol influence platelet fatty acids in the exclusively formula-fed piglet

Platelet lipid composition is important to normal platelet morphology and function, and is influenced by dietary fatty acids and cholesterol. The fatty acid composition and cholesterol content of infant formulas differs from those of human milk, but the possible effects on platelet lipids in young infants is not known. This was studied in piglets fed from birth to 18 d of age with one of eight formulas differing in saturated fatty acid chain length, or content of 18∶1, 20∶5n−3 plus 22∶6n−3, or cholesterol. A reference group of piglets fed sow milk was also studied. Sow milk has a fatty acid composition and cholesterol content similar to that of human milk. Piglets fed formulas high in 18∶1 (34.9–40.8% wt fatty acids) and low in 16.0 (≤6.5% wt fatty acids) had lower platelet counts and greater platelet size than piglets fed sow milk (40.4% 18∶1, 30.7% 16∶0). Piglets fed formulas high in 16∶0 (27–29.6%) and 18∶1 (40–40.6%), or low in both 16∶0 (5.9–6.1%) and 18∶1 (10.8–11.2%), had similar platelet counts and size to piglets fed sow milk. Platelet phospholipid % 20∶4n−6 was lower in all the groups of piglets fed formula than in the group fed sow milk. Addition of fish oil with 20∶5n−3 plus 22∶6n−3 to the formula further decreased platelet phospholipid 20∶4n−6. Addition of cholesterol to the formula increased the platelet phospholipid % 20∶4n−6 and platelet volume.     

Low concentration of oxidized low density lipoprotein suppresses platelet reactivity <Emphasis Type="Italic">in vitro</Emphasis>: An intracellular study

The intracellular mechanisms underlying oxidized low density lipoprotein (oxLDL)-signaling pathways in platelets remain obscure and findings have been controversial. Therefore, we examined the influence of oxLDL in washed human platelets. In this study oxLDL concentration-dependently (20–100 μg/mL) inhibited platelet aggregation in human platelets stimulated by collagen (1 μg/mL) and arachidonic acid (60 μM), but not by thrombin (0.02 U/mL). The activity of oxLDL was greater at 24 h in inhibiting platelet aggregation than at 12 h. At 24 h, oxLDL concentration-dependently inhibited intracellular Ca²⁺ mobilization and thromboxane B₂ formation in human platelets stimulated by collagen. In addition, at 24 h oxLDL (40 and 80 μg/mL) significantly increased the formation of cyclic AMP, but not cyclic GMP or nitrate. In an ESR study, 24 h-oxLDL (40 μg/mL) markedly reduced the ESR signal intensity of hydroxyl radicals (OH⁻) in both collagen (2 μg/mL)-activated platelets and Fenton reaction (H₂O₂+Fe²⁺). The inhibitory effect of oxLDL may induce radical-radical termination reactions by oxLDL-derived lipid radical interactions with free radicals (such as hydroxyl radicals) released from activated platelets, with a resultant lowering of intracellular Ca²⁺ mobilization followed by inhibition of thromboxane A₂ formation, thereby leading to increased cyclic AMP formation and finally inhibited platelet aggregation. This study provides new insights concerning the effect of oxLDL in platelet aggregation.     

Presence of platelet-activating factor in pyuria in humans

The relationship between the occurrence of platelet-activating factor (PAF) and neutrophils in urine from patients with urinary tract infection was examined. PAF was detected in human pyuria, when leukocyte levels reached at least 300 cells/μL (n=45), but not in normal urine (n=12). The amount of PAF found in pyuria, measured by platelet aggregation assay, was 0.01 to 13.3 pmol/mL. A close correlation was seen between the amount of PAF present and the number of urinary leukocytes (p<0.01, r=0.70). The leukocytes in pyuria consisted almost entirely of neutrophils (96±4%, mean ±S.D.). Our findings suggest that the occurrence of PAF is associated with the accumulation of neutrophils in urine. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Comparison of lipid composition ofCandida guilliermondii grown on glucose,ethanol and methanol as the sole carbon source

The carbon and energy source for aerobically grown cultures ofCandida guilliermondii profoundly influenced the neutral lipid content and the fatty acid composition of the individual lipid components. Methanol (0.80%, w/v) grown cells cultivated at 30 C in presence of 0.025% ammonium sulfate contained 12% total lipids, 67% of which was neutral lipids. Glucose (0.74%, w/v) or ethanol (0.53%, w/v) grown cells contained 21–22% total lipids, 80% of which was neutral lipids, under the same conditions. Methanol-grown cells contained a decreased 18∶1 acid (52–54% of total fatty acids) and an increased 18∶2 acid (23–25%), as compared with glucose- or ethanol-grown cells which contained 57–66% 18∶1 acid and 8–14% 18∶2 acid, in both neutral and polar lipid fractions. The relationship between methanol metabolism and desaturation of fatty acid in yeast was discussed.     

Effect of dietary α-linolenic acid and its ratio to linoleic acid on platelet and plasma fatty acids and thrombogenesis

The effect of dietary α-linolenic acid (18∶3n−3) and its ratio to linoleic acid (18∶2n−6) on platelet and plasma phospholipid (PL) fatty acid patterns and prostanoid production were studied in normolipidemic men. The study consisted of two 42-d phases. Each was divided into a 6-d pre-experimental period, during which a mixed fat diet was fed, and two 18-d experimental periods, during which a mixture of sunflower and olive oil [low 18∶3n−3 content, high 18∶2/18∶3 ratio (LO-HI diet)], soybean oil (intermediate 18∶3n−3 content, intermediate 18∶2/18∶3 ratio), canola oil (intermediate 18∶3n−3 content, low 18∶2/18∶3 ratio) and a mixture of sunflower, olive and flax oil [high 18∶3n−3 content, low 18∶2/18∶3 ratio (HI-LO diet)] provided 77% of the fat (26% of the energy) in the diet. The 18∶3n−3 content and the 18∶2/18∶3 ratio of the experimental diets were: 0.8%, 27.4; 6.5%, 6.9; 6.6%, 3.0; and 13.4%, 2.7, respectively. There were appreciable differences in the fatty acid composition of platelet and plasma PLs. Nevertheless, 18∶1n−9, 18∶2n−6 and 18∶3n−3 levels in PL reflected the fatty acid composition of the diets, although very little 18∶3n−3 was incorporated into PL. Both the level of 18∶3n−3 in the diet and the 18∶2/18∶3 ratio were important in influencing the levels of longer chain n−3 fatty acid, especially 20∶5n−3, in platelet and plasma PL. Production of 6-keto-PGF_1α was significantly (P<0.05) higher following the HI-LO diet than the LO-HI diet although dietary fat source had no effect on bleeding time or thromboxane B₂ production. The present study showed that both the level of 18∶3n−3 in the diet and its ratio to 18∶2n−6 were important in influencing long-chain n−3 fatty acid levels in platelet and plasma PL and that prostanoid production coincided with the diet-induced differences in PL fatty acid patterns.     

Inhibitory effect of stearidonic acid (18∶4 n−3) on platelet aggregation and arachidonate oxygenation

The effect of stearidonic acid (18∶4n−3) present in fish and some plant oils, such as black currant seed oil, was studied on human platelets. When added to platelets simultaneously with collagen, arachidonic acid or endoperoxide mimetic U46619, 18∶4n−3 appeared as a weak inhibitor of platelet aggregation. In addition, 18∶4n−3 did not alter the metabolism of exogenous arachidonic acid. In contrast, when preincubated with platelets after precoating onto albumin, 18∶4n−3 inhibited platelet aggregation induced by thrombin, collagen, arachidonic acid or U46619, and was as potent as eicosapentaenoic acid (20∶5n−3) tested under similar conditions. Stearidonic acid also altered the endogenous arachidonate oxygenation stimulated by low doses of thrombin, but to a significantly lesser extent than did 20∶5n−3. It seems therefore that, in addition to competing with endogenous arachidonate metabolism, 18∶4n−3 may affect platelet aggregation by another mechanism.     

Composition of mouse peritoneal macrophage phospholipid molecular species

The individual molecular species composition of diacyl, alkylacyl and alkenylacyl glycerophospholipids was determined in mouse peritoneal macrophages. A marked deterogeneity in the relative composition (mol%) of macrophage ether and ester phospholipid individual species was noted. High concentrations of 16∶0–20∶4 were found in ether phospholipids such as alkenylacyl glycerophosphoethanolamine (GPE; 27.5 mol%) and alkylacyl glycerophosphocholine (GPC; 16.6%) as compared to mol % levels of 16∶0–20∶4 in diacyl GPE (5.7%) and diacyl GPC (8.1%), respectively. Interestingly, alkenylacyl GPE was highly enriched in 1-ether (16∶0) relative to alkylacyl GPC. The predominant diacyl molecular species in glycerophosphoinositol (GPI) and glycerophosphoserine (GPS) were 18∶0–20∶4 (59.1%) and 16∶0–18∶1 (41.1%), respectively. It is noteworthy that the level of 18∶0–20∶4 was several times higher in diacyl GPI (59.1%) than in diacyl GPS (11.1%), diacyl GPE (25.7%), and diacyl GPC (3.7%). The most abundant molecular species in diacyl GPC and diacyl GPE were 16∶0–18∶1 (29.9%) and 18∶0–20∶4 (25.7%), respectively. The abundance of 20∶4 in ether phospholipids, specifically 16∶0–20∶4 and 18∶0–20∶4, in alkylacyl GPC is significant in view of the role these antecedents play in the biosynthesis of platelet-activating factor (PAF) and 20∶4-derived eicosanoids in stimulated macrophages. The unique molecular species composition of the peritoneal macrophage distinguishes this cell type from others.     

Improved HPLC assay for lipid peroxides in human plasma using the internal standard of hydroperoxide

We have developed a sensitive reversed-phase chemiluminescence HPLC approach for simultaneous quantitative and qualitative analyses of hydroperoxides of cholesteryl ester and TC in human plasma. Standard hydroperoxides of cholesteryl ester and TG and a novel internal standard (1-tetradecanyl 3-octadecenoyloxy-5β-cholan-24-oate monohydroperoxide) (I.S.) were chemically synthesized and the standard curves confirmed to be linear throughout the calibration range (1–1000 pmol). Within-day and between-day CV were less than 7%, and the recoveries were within the range of 84–93%. With sample size minimized to 0.1 mL of plasma for each run, plasma cholesteryl ester hydroperoxide levels were 189±87 nM (mean±SD) in healthy young (22–25 yr old; n=15, male/female=6∶9) and 210 ±69 nM in healthy elderly (39–60 yr old; n=6, male/female= 3∶3). TG hydroperoxide was not detected in healthy subjects. In patients with advanced liver failure (36–67 yr old; n=4, male/female=2∶2), hydroperoxide levels of plasma cholesteryl ester and TG were 11,903±9,553 nM and 3,318±1,590 nM, respectively, indicating an involvement of lipid oxidation. Sensitive and specific monitoring of plasma lipid peroxides using the present chemiluminescence HPLC approach with the synthesized I.S. may help our understanding of chemical and pathophysiological aspects of lipid peroxidation.     

Radioimmunoassay for platelet-activating factor

A radioimmunoassay (RIA) for measurement of platelet-activating factor (PAF) was developed. At a final antiserum dilution of 1∶640, the lowest detection limit of PAF was 0.1 pmol (50 pg). The standard curve obtained was suitable for measurement of PAF in amounts ranging from 0.1 pmol to 30 pmol. The antiserum showed high specificity. Cross-reaction for lysoPAF, lysophosphatidylcholine and long-chain phosphati-dylcholines was very low (less than 0.025%). 1-Palmitoyl-2-acetyl-sn-glycero-3-phosphocholine cross-reacted slightly (6.25%). PAF exogenously added to macrophage suspensions was quantitatively determined by RIA after solvent extraction and high-performance liquid chromatographic separation. RIA was also used to estimate PAF formation after stimulation of rabbit alveolar macrophages in suspension with calcium ionophore A23187.