Pharmacokinetics of soy-derived lysophosphatidylcholine compared with that of glycerophosphocholine: a randomized controlled trial.

Lysophosphatidylcholine (LPC) is present in various foods and contains a choline moiety such as in glycerophosphocholine (GPC). However, the potential of LPC as a choline source remains unclear. This study investigated the single-dose pharmacokinetics of 480 mg soy-derived LPC in 12 healthy men compared with that of either soy oil with the same lipid amount (placebo) or GPC with the same choline amount. Both LPC and GPC supplementation increased plasma choline, serum phospholipid, and serum triglyceride concentrations, but neither of them significantly elevated plasma trimethylamine N-oxide concentration. In addition, although the intake of LPC slightly increased plasma LPC16:0, LPC18:2, and total LPC concentrations, their concentrations remained within physiological ranges. No adverse events were attributed to the LPC supplementation. To the best of our knowledge, this study is the first to compare LPC and GPC pharmacokinetics in humans and shows that LPC can be a source of choline.

Association of L-α Glycerylphosphorylcholine With Subsequent Stroke Risk After 10 Years.

Importance: L-α glycerylphosphorylcholine (α-GPC, choline alphoscerate) is used globally by individuals older than 50 years based on its potential function as a precursor of acetylcholine. However, choline has previously been linked to a higher risk of cardiovascular disease via trimethylamine-N-oxide, a metabolite of choline by microbiota. Objective: To investigate the association between α-GPC use and subsequent 10-year stroke risk. Design, Setting, and Participants: A population-based, retrospective cohort study was conducted using data from the National Health Insurance Service of South Korea. Participants included men and women aged 50 years or older without underlying stroke or Alzheimer disease (N = 12 008 977). Main Outcomes and Measures: All participants were divided into whether they were prescribed α-GPC during 2006-2008. α-GPC users were matched with nonusers for all covariates to create a matched cohort. α-GPC use was further divided into durations less than 2, 2 to 6, 6 to 12, and more than 12 months of α-GPC prescriptions. The adjusted hazard ratios (aHRs) and 95% CIs for total stroke, ischemic stroke, and hemorrhagic stroke from January 1, 2009, to January 31, 2018, were calculated by multivariate Cox proportional hazards regression. Results: A total of 12 008 977 individuals (6 401 965 [53.3%] women) aged 50 years or older were included in the study. The mean (SD) age was 61.6 (9.4) years for nonusers and 68.3 (10.0) years for users, and that of the matching cohort was 68.2 (9.9) years for both groups. Compared with α-GPC nonusers (n = 11 900 100), users (n = 108 877) had a higher risk for total stroke (aHR, 1.46; 95% CI, 1.43-1.48), ischemic stroke (aHR 1.36; 95% CI, 1.33-1.39), and hemorrhagic stroke (aHR, 1.36; 95% CI, 1.28-1.44). After matching for all covariates, α-GPC users had a higher risk for total stroke (aHR, 1.43; 95% CI, 1.41-1.46), ischemic stroke (aHR, 1.34; 95% CI, 1.31-1.37), and hemorrhagic stroke (aHR, 1.37; 95% CI, 1.29-1.46). Increasing intake of α-GPC was associated with a higher risk for total stroke in a dose-response manner. Conclusions and Relevance: In this cohort study, use of α-GPC was associated with a higher 10-year incident stroke risk in a dose-response manner after adjusting for traditional cerebrovascular risk factors. Future studies are needed to determine the possible mechanisms behind the potential cerebrovascular risk-elevating effects of α-GPC.

Role of Magnetic Resonance Spectroscopy in Evaluation of Cerebral Metabolic Status Before and After Carotid Endarterectomy/Thromboendarterectomy and Carotid Artery Stenting in Patients with Asymptomatic Critical Internal Carotid Artery Stenosis.

BACKGROUND The relative efficacy of carotid endarterectomy (CEA)/thromboendarterectomy (TEA) and carotid artery stenting (CAS) already has been compared in randomized controlled trials and a meta-analysis, but only limited data exist describing the status of cerebral metabolism before and after these interventions. The aim of the present study was to compare metabolic changes before and after treatment of carotid stenosis and assess their potential clinical implications.   MATERIAL AND METHODS Patients with asymptomatic unilateral critical internal CAS were imaged with proton 3T magnetic resonance spectroscopy (H-MRS) because the technique is more sensitive than regular magnetic resonance imaging for detection of the early signs of ischemic events. Abnormal metabolite ratios detected with H-MRS may precede actual morphological changes associated with hypoperfusion as well as reperfusion changes. Ipsilateral and contralateral middle cerebral artery vascular territories were both evaluated before and after vascular intervention. H-MRS was performed within 24 h before and after surgery. Correlations in the metabolic data from H-MRS for N-acetylaspartic acid (NAA)+N-acetylaspartylglutamate, creatinine (Cr)+phosphocreatinine, and phosphocholine+glycerophosphocholine (Cho) were sought. RESULTS H-MRS voxels from 11 subjects were analyzed. Values for dCho/CrI, dCho/CrC and Cho/Naal (P<0.001) were significantly higher ipsilaterally than contralaterally. Ratios for dNaa/ChoC and Cho/NaaC were significantly higher on the non-operated side (P<0.001). CONCLUSIONS H-MRS may be helpful for assessment of patients with CAS, particularly because unlike other modalities, it reveals postoperative changes in metabolic brain status. Initial results indicate the important role of perioperative neuroprotective treatment.

Analysis of ether glycerophosphocholines at the level of C[double bond, length as m-dash]C locations from human plasma.

Plasmanyl and plasmenyl glycerophosphocholine are ether lipids featuring the 1-O-alkyl or 1-O-alk-1'-enyl ether linkage at the sn-1 position of the glycerol backbone, respectively. Aberrant levels of ether glycerophosphocholines (ether PCs) have been correlated with cellular dysfunctions and various human diseases. Profiling ether PCs with accurate structural information is challenging because of the common presence of isomeric and isobaric species in a lipidome. The Paternò-Büchi (PB) reaction, a double bond (C[double bond, length as m-dash]C) specific derivatization method, is capable of pinpointing C[double bond, length as m-dash]C locations in unsaturated lipids, when coupled with subsequent tandem mass spectrometry (MS/MS). In this study, we have tailored the acetone PB reaction for the analysis of ether PCs. PB-MS/MS via low energy collision-induced dissociation (CID) provides diagnostic ions specific to the alkenyl ether C[double bond, length as m-dash]C bond, which are different from those derived from the isolated C[double bond, length as m-dash]C bond in the alkyl or acyl chain, thereby facilitating the distinction of isomeric plasmenyl from plasmanyl PCs. PB-MS/MS coupled with high resolution MS and multi-stage MS/MS further enable confident identification of isomeric ether PCs and isobaric diacyl PCs from mixtures. A total of 45 ether PCs in human plasma have been identified for ether linkage type and chain composition, while 28 ether PCs have structures being fully characterized down to C[double bond, length as m-dash]C locations.

Formulation and bioequivalence studies of choline alfoscerate tablet comparing with soft gelatin capsule in healthy male volunteers.

PURPOSE: The aim of this study was to develop a tablet formulation of choline alfoscerate and to assess its bioequivalence by comparing its pharmacokinetic parameters with those of a commercially available soft gelatin capsule (Gliatilin®) in healthy Korean male volunteers. MATERIALS AND METHODS: Film-coated tablet formulation was optimized to control the hygroscopicity of choline alfoscerate. Bioequivalence study was performed under fasted condition with a randomized, single-dose, two-period crossover design. Subjects were orally treated with 1,200 mg of test or reference choline alfoscerate (400 mg × three doses) formulation. Blood samples were collected up to 12 hours the day before dosing to correct the baseline level of choline and 12 hours after dosing to obtain drug absorption profile. Pharmacokinetic parameters were determined after analyzing plasma concentration of choline by using LC-MS/MS. RESULTS: Hygroscopicity of choline alfoscerate was successfully controlled by adding suitable amount of Neusilin® (magnesium aluminometasilicate) in the film-coated tablet. Stability of the tablet formulation was also confirmed under the accelerated condition for 3 months. Bioequivalence study showed that the mean area under the plasma concentration-time curve from time 0 to infinity of test tablet and reference soft capsule was 3.428±2.170 and 3.305±1.803 µg⋅h/mL, respectively; the mean Cmax was 0.365±0.158 and 0.380±0.108 µg/mL, respectively; and the mean Tmax was 3.51±2.57 and 3.85±3.19 hours, respectively. The 90% CIs for geometric mean ratios of test to reference formulation for AUC0-t and Cmax were 84.51%-111.98% and 83.31%-104.10%, respectively, and satisfied the EMA regulatory criteria for bioequivalence. CONCLUSION: Pharmacokinetic parameters including the Cmax and AUC0-t determined after oral administration of the two formulations in healthy Korean male volunteers showed that the differences between the formulations (tablet vs soft capsule) were not significant for bioequivalence. Both formulations were well tolerated, with no serious adverse events reported.

Oxidized glycerophosphatidylcholines in diabetes through non-targeted metabolomics: Their annotation and biological meaning.

Lipid oxidation is one of the most important processes occurring in living cells and has been investigated through stable end-products. Currently, new insights into many physiological and pathophysiological processes provide a measurement of the first products of oxidation, e.g., oxidized glycerophosphatidylcholines (oxGPCs). Here, we evaluate the capacity of untargeted global metabolomics to measure oxGPCs in serum samples. This evaluation covered analytical reproducibility and data quality as well as the ability to capture metabolic alterations in diverse conditions. The analytical evaluation was performed based on the quality control samples, while the comparative analysis was based on the model of the development of type 2 diabetes mellitus (T2DM). The novelty of this approach arises not only from the measurement of oxGPCs instead of lipid peroxide-derived aldehydes but also from the stratification of the patients according to body mass index (BMI). Such a scenario was dictated by the fact that, despite the well-known relationship between obesity and T2DM development, there are lean individuals suffering from T2DM as well as obese people with normal glucose homeostasis. Our results provided evidence to support the ability of nontargeted metabolomics to measure oxGPCs. Comparative analysis of measured oxGPCs revealed differences in the level of oxGPCs either between different stages of disease development (insulin resistance, prediabetes) or BMI groups (normal weight, overweight, obese). The obtained results provided new insights into the metabolic processes leading to the development of T2DM and opened new paths in the investigation of the impact of body mass in T2DM progress.

Modified insoluble dietary fibers in okara affect body composition, serum metabolic properties, and fatty acid profiles in mice fed high-fat diets: an NMR investigation.

The potential health benefit of dietary fiber has attracted considerable attention in recent decades. In this study, the effects of modified dietary fibers (MDF) derived from okara on body composition, fat distribution, serum metabolomic parameters, and fatty acid profiles in mice fed high-fat diets (HFD) were evaluated by nuclear magnetic resonance (NMR)-based metabolic approach. HFD-induced C57BL mice were fed with a diet containing 100 g/kg MDF for 12 weeks. Compared with control mice, MDF-fed mice exhibited less fat and lower body weights, altered serum metabolomic profiles, and distinct fatty acid profiles. The levels of choline, phosphatidylcholine, glycerophosphorylcholine, glucose, lysine, scyllo-inositol, and glutamate for MDF group were higher than those for both CONT and HFD groups. A remarkable reduction of total cholesterol, total triglycerides, ω-6 fatty acids, alanine, citrate, creatine, or succinate was also observable for MDF group compared with HFD group. These findings demonstrated that the intake of MDF derived from okara clearly ameliorated some of the HFD-induced adverse metabolic effects and prevented adipose tissue accumulation.

Lipidomic analysis reveals the significant increase in diacylglycerophosphocholines in umbilical cord blood from pregnant women with gestational hypercholesterolemia.

Gestational hypercholesterolemia has been recognized as a risk factor of some pregnancy complications. We supposed that maternal hypercholesterolemia modified the lipid profile of the fetus. Thirty pregnant women with hypercholesterolemia and matched controls were recruited and cord blood was sampled. Lipidomic analysis was used to evaluate the lipid profile change between the two groups. The results showed that the content of diacylglycerophosphocholines (PC) was significantly high in cord blood from hypercholesterolemic pregnant women. PC (16:0/20:4) and PC (18:0/20:4) were selected as the most important lipid species in cord plasma and their contents were positively related to the total cholesterol and high-density lipoprotein cholesterol levels in cord blood. The contents of these two PCs were significantly higher in the hypercholesterolemic group than in the control group. These results suggested that gestational hypercholesterolemia might program the phospholipid metabolism in offspring.

Variation of betaine, N,N-dimethylglycine, choline, glycerophosphorylcholine, taurine and trimethylamine-N-oxide in the plasma and urine of overweight people with type 2 diabetes over a two-year period.

BACKGROUND: Plasma betaine concentrations and urinary betaine excretions have high test-retest reliability. Abnormal betaine excretion is common in diabetes. We aimed to confirm the individuality of plasma betaine and urinary betaine excretion in an overweight population with type 2 diabetes and compare this with the individuality of other osmolytes, one-carbon metabolites and trimethylamine-N-oxide (TMAO), thus assessing their potential usefulness as disease markers. METHODS: Urine and plasma were collected from overweight subjects with type 2 diabetes at four time points over a two-year period. We measured the concentrations of the osmolytes: betaine, glycerophosphorylcholine (GPC) and taurine, as well as TMAO, and the one-carbon metabolites, N,N-dimethylglycine (DMG) and free choline. Samples were measured using tandem mass spectrometry (LC-MS/MS). RESULTS: Betaine showed a high degree of individuality (or test-retest reliability) in the plasma (index of individuality = 0.52) and urine (index of individuality = 0.45). Betaine in the plasma had positive and negative log-normal reference change values (RCVs) of 54% and -35%, respectively. The other osmolytes, taurine and GPC were more variable in the plasma of individuals compared to the urine. DMG and choline showed high individuality in the plasma and urine. TMAO was highly variable in the plasma and urine (log-normal RCVs ranging from 403% to -80% in plasma). CONCLUSIONS: Betaine is highly individual in overweight people with diabetes. Betaine, its metabolite DMG, and precursor choline showed more reliability than the osmolytes, GPC and taurine. The low reliability of TMAO suggests that a single TMAO measurement has low diagnostic value.

Lipid fingerprinting in women with early-onset preeclampsia: a first look.

OBJECTIVES: The aim of this preliminary study was to characterize the plasma lipid profiling of women with preeclampsia. DESIGN AND METHODS: Plasma samples of 8 pregnant women with early-onset preeclampsia and 8 normal pregnant women were evaluated. Lipids were extracted from plasma using the Bligh-Dyer protocol. The extracts were subjected to MALDI-MS. Data matrix was exported for partial least squares discriminant analysis (PLS-DA) and a parameter VIP was employed to reflect the variable importance in the discriminant analysis. The major discriminant variables were selected and underwent to Mann-Whitney U test. RESULTS: A total of 1290 ions were initially identified and twelve m/z signals were highlighted as the most important lipids for the discrimination of patients with preeclampsia. The identification of these differential lipids was carried out through Lipid Database Search. CONCLUSIONS: The main classes identified were glycerophosphocholines [GP01], glycerophosphoserines [GP03], glycerophosphoglycerols [GP04], glycosyldiradylglycerols [GL05] and glycerophosphates [GP10].

Profiling phospholipid elution in reversed-phase LC-MS/MS bioanalytical methods in order to avoid matrix effects.

BACKGROUND: Endogenous phospholipids have a profound matrix effect in bioanalytical LC-MS methods and considerable effort is invested in strategies to minimize their impact either by removal during sample processing or chromatographic separation during the analytical run. The aim of the research presented in this article was to investigate the latter approach, under reversed-phase conditions. RESULTS: The retention of glycerophosphocholines (GPCs) in mobile phases employing acetonitrile demonstrated a complex 'U-shaped' relationship with the percentage of organic. Conversely, in mobile phases employing methanol, the relationship between retention and percentage of organic was entirely predictive and unaffected by changes in the mobile phase pH. The GPC elution profile was also qualitatively equivalent irrespective of the species from which the plasma was derived. CONCLUSION: The predictive nature of GPC retention, under reversed-phase chromatography and with MeOH as organic modifier, is an important finding that should allow for a more streamlined and simplified strategy in the development of bioanalytical assays.

Metabotyping of long-lived mice using 1H NMR spectroscopy.

Significant advances in understanding aging have been achieved through studying model organisms with extended healthy lifespans. Employing 1H NMR spectroscopy, we characterized the plasma metabolic phenotype (metabotype) of three long-lived murine models: 30% dietary restricted (DR), insulin receptor substrate 1 null (Irs1-/-), and Ames dwarf (Prop1df/df). A panel of metabolic differences were generated for each model relative to their controls, and subsequently, the three long-lived models were compared to one another. Concentrations of mobile very low density lipoproteins, trimethylamine, and choline were significantly decreased in the plasma of all three models. Metabolites including glucose, choline, glycerophosphocholine, and various lipids were significantly reduced, while acetoacetate, d-3-hydroxybutyrate and trimethylamine-N-oxide levels were increased in DR compared to ad libitum fed controls. Plasma lipids and glycerophosphocholine were also decreased in Irs1-/- mice compared to controls, as were methionine and citrate. In contrast, high density lipoproteins and glycerophosphocholine were increased in Ames dwarf mice, as were methionine and citrate. Pairwise comparisons indicated that differences existed between the metabotypes of the different long-lived mice models. Irs1-/- mice, for example, had elevated glucose, acetate, acetone, and creatine but lower methionine relative to DR mice and Ames dwarfs. Our study identified several potential candidate biomarkers directionally altered across all three models that may be predictive of longevity but also identified differences in the metabolic signatures. This comparative approach suggests that the metabolic networks underlying lifespan extension may not be exactly the same for each model of longevity and is consistent with multifactorial control of the aging process.

Is serum or plasma more appropriate for intersubject comparisons in metabolomic studies? An assessment in patients with small-cell lung cancer.

In clinical analyses, the most appropriate biofluid should be analyzed for optimal assay performance. For biological fluids, the most readily accessible is blood, and metabolomic analyses can be performed either on plasma or serum. To determine the optimal agent for analysis, metabolic profiles of matched human serum and plasma were assessed by gas chromatography/time-of-flight mass spectrometry and ultrahigh-performance liquid chromatography mass spectrometry (in positive and negative electrospray ionization modes). Comparison of the two metabolomes, in terms of reproducibility, discriminative ability and coverage, indicated that they offered similar analytical opportunities. An analysis of the variation between 29 small-cell lung cancer (SCLC) patients revealed that the differences between individuals are markedly similar for the two biofluids. However, significant differences between the levels of some specific metabolites were identified, as were differences in the intersubject variability of some metabolite levels. Glycerophosphocholines, erythritol, creatinine, hexadecanoic acid, and glutamine in plasma, but not in serum, were shown to correlate with life expectancy for SCLC patients, indicating the utility of metabolomic analyses in clinical prognosis and the particular utility of plasma in relation to the clinical management of SCLC.

Development and validation of a liquid chromatography-mass spectrometry metabonomic platform in human plasma of liver failure caused by hepatitis B virus.

This paper presents an liquid chromatography (LC)/mass spectrometry (MS)-based metabonomic platform that combined the discovery of differential metabolites through principal component analysis (PCA) with the verification by selective multiple reaction monitoring (MRM). These methods were applied to analyze plasma samples from liver disease patients and healthy donors. LC-MS raw data (about 1000 compounds), from the plasma of liver failure patients (n = 26) and healthy controls (n = 16), were analyzed through the PCA method and a pattern recognition profile that had significant difference between liver failure patients and healthy controls (P < 0.05) was established. The profile was verified in 165 clinical subjects. The specificity and sensitivity of this model in predicting liver failure were 94.3 and 100.0%, respectively. The differential ions with m/z of 414.5, 432.0, 520.5, and 775.0 were verified to be consistent with the results from PCA by MRM mode in 40 clinical samples, and were proved not to be caused by the medicines taken by patients through rat model experiments. The compound with m/z of 520.5 was identified to be 1-Linoleoylglycerophosphocholine or 1-Linoleoylphosphatidylcholine through exact mass measurements performed using Ion Trap-Time-of-Flight MS and METLIN Metabolite Database search. In all, it was the first time to integrate metabonomic study and MRM relative quantification of differential peaks in a large number of clinical samples. Thereafter, a rat model was used to exclude drug effects on the abundance of differential ion peaks. 1-Linoleoylglycerophosphocholine or 1-Linoleoylphosphatidylcholine, a potential biomarker, was identified. The LC/MS-based metabonomic platform could be a powerful tool for the metabonomic screening of plasma biomarkers.

Reducing glycerophosphocholine lipid matrix interference effects in biological fluid assays by using high-turbulence liquid chromatography.

Matrix interferences can severely affect quantitative assays of biological samples when electrospray ionization (ESI) is employed with liquid chromatography/tandem mass spectrometry (LC/MS/MS). A major source of matrix interferences for plasma sample analyses is the presence of glycerophosphocholine (GPCho) lipids. The efficiency of online high-turbulence liquid chromatography (HTLC) extraction for eliminating these lipids is evaluated and the interfering effects of endogenous lipids on human plasma assays are measured for pharmaceutical compounds having a wide variety of chemical properties. It is found that GPCho lipids, represented by 16:0, 18:1 and 18:0 LPC (lysophosphatidylcholine) and 16:0-18:2 PC, cause variations for hydrophobic compound analyses even when optimal online HTLC extraction conditions are employed. The efficiency for lipid removal depends on the organic content of the transfer solvent, but turbulent flow loading has no significant effect.

Choline status in newborns, infants, children, breast-feeding women, breast-fed infants and human breast milk.

This study assessed the choline status in newborns, infants, children, breast-feeding women, breast milk, infant formula, breast-fed and formula-fed infants. The serum free choline level was 35.1+/-1.1 micromol/L at birth and decreased to 24.2+/-1.6, 18.1+/-0.8, 16.3+/-0.9, 14.3+/-0.8, 12.9+/-0.6 or 10.9+/-0.6 micromol/L at 22-28, 151-180, 331-365, 571-730, 731-1095 or 4016-4380 days after birth, respectively. The serum phospholipid-bound choline level was 1997+/-75 micromol/L at birth and increased gradually to 2315+/-190 or 2572 +/-100 micromol/L at 571-730 or 4016-4380 days after birth, respectively. In breast-feeding women, serum free and phospholipid-bound choline levels were doubled at 12-28 days after birth, they decreased toward the control values with time. Free choline, phosphocholine and glycerophosphocholine were major choline compounds in breast milk. Their concentrations in mature milk were much greater than in colostrum and serum. Choline contents of breast milk varied greatly between mothers, and milk free choline levels were correlated with serum free choline (r=.541; P<.001), phospholipid-bound choline (r=.527; P<.001) and glycerophosphocholine (r=.299; P<.01) concentrations and lactating days (r=.520; P<.001). In breast-fed infants, serum free choline concentrations were correlated with free choline (r=.47; P<.001), phosphocholine (r=.345; P<.002), glycerophosphocholine (r=.311; P<.01) and total choline (r=.306; P<.01) contents of breast milk. Serum free choline concentration in formula-fed infants was lower than breast-fed infants. These data show that (a) circulating choline status is elevated during infancy and lactation, (b) choline contents of breast milk vary between mothers and milk free choline contents are influenced by maternal circulating choline status, and (c) the choline contents of breast milk can influence infants' circulating choline status.

12(S)-Hydroperoxy-eicosatetraenoic acid increases arachidonic acid availability in collagen-primed platelets.

Lipid hydroperoxides have been reported to regulate cell function and eicosanoid formation. The aim of the present study was to determine the effect of 12(S)-hydroperoxy-eicosatetraenoic acid [12(S)-HPETE], the platelet 12-lipoxygenase-derived hydroperoxide of arachidonic acid (AA), on the availability of nonesterified AA, which represents a rate-limiting step in the biosynthesis of eicosanoids. The coincubation of human platelets with concentrations of 12(S)-HPETE below 50 nM and subthreshold concentrations (STC) of collagen (less than 0.24 microg/ml) significantly enhanced platelet aggregation and the formation of thromboxane B(2), the stable catabolite of the potent aggregating agent thromboxane A(2). In addition, the nonesterified endogenous AA concentration increased by 3-fold. Arachidonoyl-containing molecular species concentrations of 1,2-diacyl-glycero-3-phosphocholine, 1-alkyl-2-acyl-glycero-3-phosphocholine, and 1-alkenyl-2-acyl-glycero-3-phosphoethanolamine decreased specifically in response to 12(S)-HPETE, whereas no significant changes were observed within 1,2-diacyl-glycero-3-phosphoethanolamine and 1,2-diacyl-glycero-3-phosphoinositol molecular species. The 12(S)-HPETE-induced increase in nonesterified AA was fully prevented by arachidonoyl trifluoromethyl ketone, an inhibitor of cytosolic phospholipase A(2) (cPLA(2)), and cPLA(2) was translocated to membranes and phosphorylated in platelets incubated with 12(S)-HPETE. In conclusion, these results indicate that nanomolar concentrations of 12(S)-HPETE could play a significant role in controlling the level of endogenous AA and the formation of thromboxane, thereby potentiating platelet function.

Rapid kinetics of insertion and accessibility of spin-labeled phospholipid analogs in lipid membranes: a stopped-flow electron paramagnetic resonance approach.

Spin-labeled phospholipid analogs have been employed to probe the transbilayer distribution of endogenous phospholipids in various membrane systems. To determine the transmembrane distribution of the spin-labeled analogs, the analogs are usually inserted into the membrane of interest and subsequently the amount of analog in the outer membrane leaflet is determined either by chemical reduction with ascorbate or by back-exchange to bovine serum albumin (BSA). For accurate determination of the transbilayer distribution of analogs, both the kinetics of incorporation and those of accessibility of analogs to ascorbate or BSA have to be fast in comparison to their transbilayer movement. By means of stopped-flow electron paramagnetic resonance (EPR) spectroscopy, we have studied the kinetics of incorporation of the spin-labeled phosphatidylcholine (PC) analog 1-palmitoyl-2-(4-doxylpentanoyl)-sn-glycero-3-phosphocholine (SL-PC) and of its accessibility to chemical reduction and to back-exchange at room temperature. Incorporation of SL-PC into the outer leaflet of egg phosphatidylcholine (EPC) and red cell ghost membranes was essentially completed within 5 s. Ninety percent of the SL-PC molecules located in the outer membrane leaflet of those membranes were extracted by BSA within 15 s. All exterior-facing SL-PC molecules were reduced by ascorbate in a pseudo-first-order reaction within 60 s in EPC membranes and within 90 s in red cell ghost membranes. The rate of the reduction process could be enhanced by approximately 30-fold when 6-O-phenyl-ascorbic acid was used instead of ascorbate as the reducing agent. The results are discussed in light of assaying rapid transbilayer movement of spin-labeled analogs in biological membranes.

Transverse redistribution of phospholipids during human platelet activation: evidence for a vectorial outflux specific to aminophospholipids.

The redistribution kinetics of phospholipids during human platelet activation by calcium ionophore were investigated to determine the specificity of the observed phospholipid outflux [Bassé et al. (1993) Biochemistry 32, 2337]. (1) Two double-labeling experiments were performed with a combination of equal amounts of spin- and fluorescently-labeled phosphatidylserine and phosphatidylcholine. During A23187-induced activation, 50% of the internal phosphatidylserine analogs were rapidly (t1/2 < 1 min) reexposed on the platelet surface while no reciprocal influx of the external phosphatidylcholine analogs was observed. (2) Treatment with chlorpromazine allowed the internalization of 20% of external spin-labeled sphingomyelin or spin-labeled phosphatidylcholine, without either inducing platelet activation or interfering with aminophospholipid translocase activity or with A23187-induced activation (dense granule secretion and vesicle shedding). During A23187-induced activation, none of the previously internalized choline head phospholipids were exposed externally, while spin-labeled phosphatidylserine outward movements were similar irrespective of whether platelets were pretreated or not pretreated with chlorpromazine. Our results demonstrated that during strong platelet activation (1) the PL excess in the internal leaflet, due to the probe addition, is not responsible for their outflux; (2) the rapid aminophospholipid outflux is definitely a vectorial outflux not counterbalanced by a rapid reciprocal influx of choline head phospholipids (i.e., not scrambling); and (3) the vectorial outflux is specific for aminophospholipids since previously internalized sphingomyelin and phosphatidylcholine did not move outward. This suggests that the specific vectorial outflux of aminophospholipids could be catalyzed by a "reverse aminophospholipid translocase" activity.

Absorption, tissue distribution and excretion of radiolabelled compounds in rats after administration of [14C]-L-alpha-glycerylphosphorylcholine.

The kinetics and metabolism of L-alpha-glycerylphosphoryl-choline (alpha-GPC) were investigated in male and female rats after i.v. (10 mg/kg) and oral doses (100-300 mg/kg). alpha-GPC was labelled with [14C]-glycerol ([14G]-GPC) or [14C]-choline ([14C]-GPC). Different kinetic and metabolic profiles were observed after i.v. and oral administration. It is assumed that alpha-GPC is hydrolyzed by phosphodiesterases in the gut mucosa. The different labelled metabolites have different kinetic properties of absorption, distribution and clearance, leading to different blood concentration-time curves of total radioactivity. Both labelled compounds gave a wide distribution of radioactivity, particularly concentrated in the liver, kidney, lung and spleen compared to blood. Brain concentrations of [14C]-GPC were comparable to ([14G]-GPC) or lower than ([14C]-GPC) total blood radioactivity. The metabolite profile in the perfused brain showed a small amount of choline and two unknown metabolites, probably the same as in blood. In addition, choline was incorporated into brain phospholipids in increasing amounts within 24 h of dosing. In all cases renal and fecal excretion of radioactivity was low and comparable for [14G]-GPC and [14C]-GPC. Mostly the administered radioactivity was exhaled as 14CO2, this degradation being faster and more pronounced for the glycerol-labelled metabolites than for the choline-labelled metabolites for both routes of administration. In all cases the results were the same for male and female rats.