Quantitation of apolipoprotein B-48 in triacylglycerol-rich lipoproteins by a specific enzyme-linked immunosorbent assay

This paper describes the use of an antiserum, specific for apolipoprotein (apo) B-48, in a competitive, enzyme-linked immunosorbent assay (ELISA) for apo B-48 in triacylglycerol-rich lipoprotein (TRL) fractions prepared from fasting and post-prandial plasma samples. Previously we showed the antiserum to act as an effective immunoblotting agent following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its use in this ELISA indicates that the antiserum recognises the C-terminal region of the protein on the surface of lipoprotein particles. The ELISA had a sensitivity of less than 37 ng/ml and intra- and inter-assay coefficients of variation of 3.8% and 8.6%, respectively. There was no cross-reaction in the ELISA against serum albumin, ovalbumin, thyroglobulin, or apo B-100 (purified by immunoaffinity chromatography), and high lipid concentrations (as Intralipid) did not interfere. A low density lipoprotein fraction reacted in the ELISA but SDS-PAGE-Western blot analysis confirmed the presence, in the fraction, of a small amount of apo B-48, indicating the existence of low density dietary-derived lipoprotein particles. ELISA and SDS-PAGE-Western blot analysis were used to measure apo B-48 in 12 series of postprandial samples collected from 4 diabetic and 8 normal subjects, following test meals of varying fat content. The mean correlation between the two methods was r = 0.74. The mean fasting concentration of apo B-48 in the TRL fractions from 15 healthy men was 0.46 μg/ml of plasma.

The effect of triacylglycerol fatty acid positional distribution on postprandial plasma metabolite and hormone responses in normal adult men

The present study has examined the possibility that the positional distribution of fatty acids on dietary triacyglycerol (TAG) influences the postprandial response to a liquid meal in adult subjects. Postprandial TAG, non-esterified fatty acids (NEFA), ketones, glucose, insulin and gastric inhibitory polypeptide (GIP) responses were monitored in sixteen normal adult male subjects over 6 h following consumption of test meals containing dietary TAG in which palmitic acid was predominantly on the sn-1 (Control) or sn-2 positions (Betapol). Plasma total TAG, chylomicron-rich TAG and chylomicron-poor TAG concentrations were identical in response to the two test meals. The peak increase (mean (SD)) in chylomicron TAG was 0.85 (0.46) mmol/l after the Control meal and 0.85 (0.42) mmol/l after the Betapol meal. Plasma glucose, insulin, GIP, NEFA and ketone concentrations were also very similar following the two meals. It is concluded that dietary TAG containing saturated fatty acids on the sn-2 position appear in plasma at a similar level and over a similar timescale to TAG in which saturated fatty acids are predominantly located on sn-1 or sn-3 positions. The results reported in the present study demonstrate that the positional distribution of fatty acids on dietary TAG is not an important determinant of postprandial lipaemia in adult male subjects, but do not exclude the possibility that different responses may occur when these dietary TAG are given long term.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase, endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Effects of chronic consumption of fruit and vegetable puree-based drinks on vasodilation, plasma oxidative stability and antioxidant status

Background: Fruit and vegetable-rich diets are associated with a reduced cardiovascular disease (CVD) risk. This protective effect may be a result of the phytochemicals present within fruits and vegetables (F&V). However, there can be considerable variation in the content of phytochemical composition of whole F&V depending on growing location, cultivar, season and agricultural practices, etc. Therefore, the present study investigated the effects of consuming fruits and vegetables as puree-based drinks (FVPD) daily on vasodilation, phytochemical bioavailability, antioxidant status and other CVD risk factors. FVPD was chosen to provide a standardised source of F&V material that could be delivered from the same batch to all subjects during each treatment arm of the study. Methods: Thirty-nine subjects completed the randomised, controlled, cross-over dietary intervention. Subjects were randomised to consume 200 mL of FVPD (or fruit-flavoured control), daily for 6 weeks with an 8-week washout period between treatments. Dietary intake was measured using two 5-day diet records during each cross-over arm of the study. Blood and urine samples were collected before and after each intervention and vasodilation assessed in 19 subjects using laser Doppler imaging with iontophoresis. Results: FVPD significantly increased dietary vitamin C and carotenoids (P < 0.001), and concomitantly increased plasma α- and β-carotene (P < 0.001) with a near-significant increase in endothelium-dependent vasodilation (P = 0.060). Conclusions: Overall, the findings obtained in the present study showed that FVPD were a useful vehicle to increase fruit and vegetable intake, significantly increasing dietary and plasma phytochemical concentrations with a trend towards increased endothelium-dependent vasodilation.

A period 2 genetic variant interacts with plasma SFA to modify plasma lipid concentrations in adults with metabolic syndrome

Genetic variants of Period 2 (PER2), a circadian clock gene, have been linked to metabolic syndrome (MetS). However, it is still unknown whether these genetic variants interact with the various types of plasma fatty acids. This study investigated whether common single nucleotide polymorphisms (SNPs) in the PER2 locus (rs934945 and rs2304672) interact with various classes of plasma fatty acids to modulate plasma lipid metabolism in 381 participants with MetS in the European LIPGENE study. Interestingly, the rs2304672 SNP interacted with plasma total SFA concentrations to affect fasting plasma TG, TG-rich lipoprotein (TRL-TG), total cholesterol, apoC-II, apoB, and apoB-48 concentrations (P-interaction < 0.001–0.046). Carriers of the minor allele (GC+GG) with the highest SFA concentration (>median) had a higher plasma TG concentration (P = 0.001) and higher TRL-TG (P < 0.001) than the CC genotype. In addition, participants carrying the minor G allele for rs2304672 SNP and with a higher SFA concentration (>median) had higher plasma concentrations of apo C-II (P < 0.001), apo C-III (P = 0.009), and apoB-48 (P = 0.028) compared with the homozygotes for the major allele (CC). In summary, the rs2304672 polymorphism in the PER2 gene locus may influence lipid metabolism by interacting with the plasma total SFA concentration in participants with MetS. The understanding of these gene-nutrient interactions could help to provide a better knowledge of the pathogenesis in MetS.

Transient heparin-induced platelet activation linked to generation of platelet 12-lipoxygenase: findings from a randomised controlled trial

Previously we demonstrated that heparin administration during carotid endarterectomy (CEA) caused a marked, but transient increase in platelet aggregation to arachidonic acid (AA) and adenosine diphosphate (ADP), despite effective platelet cyclo-oxygenase-1 (COX-1) inhibition with aspirin. Here we investigated the metabolism of AA via platelet 12-lipoxygenase (12-LOX) as a possible mediator of the observed transient aspirin resistance, and compared the effects of unfractionated (UFH) and low-molecular-weight (LMWH) heparin. A total of 43 aspirinated patients undergoing CEA were randomised in the trial to 5,000 IU UFH (n=22) or 2,500 IU LMWH (dalteparin, n=21). Platelet aggregation to AA (4x10⁻³) and ADP (3x10⁻⁶) was determined, and the products of the COX-1 and 12-LOX pathways; thromboxane B₂ (TXB₂) and 12-hydroxyeicosatretraenoic acid (12-HETE) were measured in plasma, and in material released from aggregating platelets.Aggregation to AA increased significantly (~10-fold) following heparinisation (p<0.0001), irrespective of heparin type (p=0.33). Significant, but smaller (~2-fold) increases in aggregation to ADP were also seen, which were significantly lower in the platelets of patients randomised to LMWH (p<0.0001). Plasma levels of TxB2 did not rise following heparinisation (p=0.93), but 12-HETE increased significantly in the patients' plasma, and released from platelets stimulated in vitro withADP, with both heparin types (p<0.0001). The magnitude of aggregation to ADP correlated with 12-HETE generation (p=0.03). Heparin administration during CEA generates AA that is metabolised to 12-HETE via the 12-LOX pathway, possibly explaining the phenomenon of transient heparin-induced platelet activation. LMWH has less effect on aggregation and 12-HETE generation than UFH when the platelets are stimulated with ADP.

Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

Flavonoid inhibitory pharmacodynamics on platelet function in physiological environments

The complex relationship between flavonoid-based nutrition and cardiovascular disease may be dissected by understanding the activities of these compounds in biological systems. The aim of the present study was to explore a hierarchy for the importance of dietary flavonoids on cardiovascular health by examining the structural basis for inhibitory effects of common, dietary flavonoids (quercetin, apigenin, and naringenin) and the plasma metabolite, tamarixetin. Understanding flavonoid effects on platelets in vivo can be informed by investigations of the ability of these compounds to attenuate the function of these cells. Inhibition of platelet function in whole blood and plasma was structure-dependent. The order of potency was apigenin > tamarixetin > quercetin = naringenin indicating that in vivo, important functional groups are potentially a methylated B ring, and a non-hydroxylated, planar C ring. Apigenin and the methylated metabolite of quercetin, tamarixetin significantly reduced thrombus volume at concentrations (5 μM) that suggested their reported physiological levels (0.1-1 μM) may exert low levels of inhibition. Flavonoid interactions with erythrocytes, leukocytes and human serum albumin in whole blood reduce their inhibitory activities against platelet function. The diminished inhibitory activity of flavonoids that we observed in whole blood and plasma indicated that these interactions do not overcome the attenuating effects of these compounds. Furthermore, inhibition of platelet aggregation by flavonoids was enhanced with increases in exposure time, indicating the potential for measurable inhibitory effects during resident plasma times. We conclude that flavonoid structures may be a major influence of their activities in vivo with methylated metabolites and those of flavones being more potent than those of flavonols and flavanones.

Flavonoid-rich fruits and vegetables improve microvascular reactivity and inflammatory status in men at risk of cardiovascular disease – FLAVURS: a randomized controlled trial

BACKGROUND: Observed associations between increased fruit and vegetable (F&V) consumption, particularly those F&Vs that are rich in flavonoids, and vascular health improvements require confirmation in adequately powered randomized controlled trials. OBJECTIVE: This study was designed to measure the dose-response relation between high-flavonoid (HF), low-flavonoid (LF), and habitual F&V intakes and vascular function and other cardiovascular disease (CVD) risk indicators. DESIGN: A single-blind, dose-dependent, parallel randomized controlled dietary intervention study was conducted. Male and female low-F&V consumers who had a ≥1.5-fold increased risk of CVD (n = 174) were randomly assigned to receive an HF F&V, an LF F&V, or a habitual diet, with HF and LF F&V amounts sequentially increasing by 2, 4, and 6 (+2, +4, and +6) portions/d every 6 wk over habitual intakes. Microvascular reactivity (laser Doppler imaging with iontophoresis), arterial stiffness [pulse wave velocity, pulse wave analysis (PWA)], 24-h ambulatory blood pressure, and biomarkers of nitric oxide (NO), vascular function, and inflammation were determined at baseline and at 6, 12, and 18 wk. RESULTS: In men, the HF F&V diet increased endothelium-dependent microvascular reactivity (P = 0.017) with +2 portions/d (at 6 wk) and reduced C-reactive protein (P = 0.001), E-selectin (P = 0.0005), and vascular cell adhesion molecule (P = 0.0468) with +4 portions/d (at 12 wk). HF F&Vs increased plasma NO (P = 0.0243) with +4 portions/d (at 12 wk) in the group as a whole. An increase in F&Vs, regardless of flavonoid content in the groups as a whole, mitigated increases in vascular stiffness measured by PWA (P = 0.0065) and reductions in NO (P = 0.0299) in the control group. CONCLUSION: These data support recommendations to increase F&V intake to ≥6 portions daily, with additional benefit from F&Vs that are rich in flavonoids, particularly in men with an increased risk of CVD. This trial was registered at www.controlled-trials.com as ISRCTN47748735.

Apolipoprotein e genotype has a modest impact on the postprandial plasma response to meals of varying fat composition in healthy men in a randomized controlled trial

BACKGROUND:Apolioprotein E (APOE) genotype is reported to influence a person's fasting lipid profile and potentially the response to dietary fat manipulation. The impact of APOE genotype on the responsiveness to meals of varying fat composition is unknown. OBJECTIVE:We examined the effect of meals containing 50 g of fat rich in saturated fatty acids (SFAs), unsaturated fatty acids (UNSATs), or SFAs with fish oil (SFA-FO) on postprandial lipemia. METHOD:A randomized, controlled, test meal study was performed in men recruited according to the APOE genotype (n = 10 APOE3/3, n = 11 APOE3/E4). RESULTS:For the serum apoE response (meal × genotype interaction P = 0.038), concentrations were on average 8% lower after the UNSAT than the SFA-FO meal in APOE4 carriers (P = 0.015) only. In the genotype groups combined, there was a delay in the time to reach maximum triacylglycerol (TG) concentration (mean ± SEM: 313 ± 25 vs. 266 ± 27 min) and higher maximum nonesterified fatty acid (0.73 ± 0.05 vs. 0.60 ± 0.03 mmol/L) and glucose (7.92 ± 0.22 vs. 7.25 ± 0.22 mmol/L) concentrations after the SFA than the UNSAT meal, respectively (P ≤ 0.05). In the Svedberg flotation rate 60-400 TG-rich lipoprotein fraction, meal × genotype interactions were observed for incremental area under the curve (IAUC) for the TG (P = 0.038) and apoE (P = 0.016) responses with a 58% lower apoE IAUC after the UNSAT than the SFA meal (P = 0.017) in the E4 carriers. CONCLUSIONS:Our data indicate that APOE genotype had a modest impact on the postprandial response to meals of varying fat composition in normolipidemic men. The physiologic importance of greater apoE concentrations after the SFA-rich meals in APOE4 carriers may reflect an impact on TG-rich lipoprotein clearance from the circulation. This trial was registered at clinicaltrials.gov as NCT01522482.

EISCAT/CRRES observations: nightside ionospheric ion outflow and oxygen-rich substorm injections

We present combined observations made near midnight by the EISCAT radar, all-sky cameras and the combined released and radiation efects satellite (CRRES) shortly before and during a substorm. In particular, we study a discrete, equatorward-drifting auroral arc, seen several degrees poleward of the onset region. The arc passes through the field-aligned beam of the EISCAT radar and is seen to be associated with a considerable upflow of ionospheric plasma. During the substorm, the CRRES satellite observed two major injections, 17 min apart, the second of which was dominated by O+ ions. We show that the observed are was in a suitable location in both latitude and MLT to have fed O+ ions into the second injection and that the upward flux of ions associated with it was sufficient to explain the observed injection. We interpret these data as showing that arcs in the nightside plasma-sheet boundary layer could be the source of O+ ions energised by a dipolarisation of the mid- and near-Earth tail, as opposed to ions ejected from the dayside ionosphere in the cleft ion fountain.

Carotenoids but not flavonoids are associated with improvements in spatial working memory in younger adults in a flavonoid-rich v. -poor fruit and vegetable intervention study

Findings from animal studies suggest that components of fruit and vegetables (F&V) may protect against, and even reverse, age-related decline(1,2) in aspects of cognitive functioning such as spatial working memory (SWM). Human subjects in vivo and in vitro studies indicate that anti-inflammatory, anti-oxidant and cell-signalling properties of flavonoids and carotenoids, non-nutrient components of F&V, may underpin this protective effect(3–5). The Flavonoid University of Reading Study (FLAVURS), designed to explore the dose-response relationship between dietary F&V flavonoids and CVD, enabled the investigation of such an association with SWM. FLAVURS is an 18-week parallel three-arm randomised controlled dietary intervention trial with four time points, measured at 6-weekly intervals from baseline. Low F&V consumers at risk of CVD aged 26–70 years were randomly assigned to high flavonoid (HF), low flavonoid (LF) or control group. F&V intake increased by two daily 80 g portions every 6 weeks, with either HF or LF F&V, in addition to each participant's habitual diet, while controls maintained their habitual diet. At each visit, participants completed a cognitive test battery with SWM as the primary outcome. The HF group showed significantly higher levels of urinary flavonoids than LF or controls at 12 weeks (P<0.001) as expected, but surprisingly only higher levels than LF at 18 weeks (P<0.01). The LF group showed higher levels of plasma carotenoids than the other groups at 18 weeks (P<0.001). No group differences were found for SWM overall, however, age-group sub-analyses (26–50 and 51–70 years of age) showed differences from 0 to 18 weeks for younger adults, with LF improving significantly more than the other two groups on SWM (P<0.05). As nutritional absorption is known to decrease with age, separate stepwise regressions were performed on the two age groups irrespective of dietary group, with urinary flavonoids and plasma carotenoids as predictors. For younger adults, improved SWM performance from 0 to 18 weeks was associated with higher carotenoid levels, β=0.28, t(55)=2.10, P<0.05, accounting for 7.5% of the variance, R2=0.075, F(1,54)=4.41, P=0.040. For older adults, no between-group SWM differences were found. Findings suggest that F&V-based flavonoids and carotenoids may provide benefits for cognitive function, and that carotenoids in particular may improve cognitive performance in SWM. Given that these benefits were restricted to younger adults, future work is needed to test the reliability of this finding, as well as determine the mechanisms by which age-dependent differences in F&V responsiveness occur.

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.

Interaction between BMI and APOE genotype is associated with changes in the plasma long-chain-PUFA response to a fish-oil supplement in healthy participants

BACKGROUND: Carriers of the apolipoprotein E ɛ4 (APOE4) allele are lower responders to a docosahexaenoic acid (DHA) supplement than are noncarriers. This effect could be exacerbated in overweight individuals because DHA metabolism changes according to body mass index (BMI; in kg/m²). OBJECTIVES: We evaluated the plasma fatty acid (FA) response to a DHA-rich supplement in APOE4 carriers and noncarriers consuming a high-saturated fat diet (HSF diet) and, in addition, evaluated whether being overweight changed this response. DESIGN: This study was part of the SATgenɛ trial. Forty-one APOE4 carriers and 41 noncarriers were prospectively recruited and consumed an HSF diet for 8-wk followed by 8 wk of consumption of an HSF diet with the addition of DHA and eicosapentaenoic acid (EPA) (HSF + DHA diet; 3.45 g DHA/d and 0.5 g EPA/d). Fasting plasma samples were collected at the end of each intervention diet. Plasma total lipids (TLs) were separated into free FAs, neutral lipids (NLs), and phospholipids by using solid-phase extraction, and FA profiles in each lipid class were quantified by using gas chromatography. RESULTS: Because the plasma FA response to the HSF + DHA diet was correlated with BMI in APOE4 carriers but not in noncarriers, the following 2 groups were formed according to the BMI median: low BMI (<25.5) and high BMI (≥25.5). In response to the HSF + DHA diet, there were significant BMI × genotype interactions for changes in plasma concentrations of arachidonic acid and DHA in phospholipids and TLs and of EPA in NLs and TLs (P ≤ 0.05). APOE4 carriers were lower plasma responders to the DHA supplement than were noncarriers but only in the high-BMI group. CONCLUSIONS: Our findings indicate that apolipoprotein E genotype and BMI may be important variables that determine the plasma long-chain PUFA response to dietary fat manipulation. APOE4 carriers with BMI ≥25.5 may need higher intakes of DHA for cardiovascular or other health benefits than do noncarriers

Intracellular trafficking, localization, and mobilization of platelet-borne thiol isomerases

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13dJinx mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.