Lipophilic Radical Initiator Research Articles

Plant derived ingredients rich in nitrates or phenolics for protection of pork against protein oxidation

A pork model system containg phenolic extracts (citrus, rosemary, and acerola), traditional Spanish food ingredients (paprika, garlic, and oregano), or natural nitrate sources (beet, lettuce, arugula, spinach, chard, celery, and watercress) were oxidized by an hydrophilic (OXHydro, 2,2'-azobis(2-amidinopropane)-dihydrochloride; AAPH) or lipophilic (OXLip, 2,2'-azobis(2,4-dimethylvaleronitrile; AMVN) radical initiator. Citrus as well as lettuce and spinach protected almost fully against protein thiol loss and showed efficient radical scavenging activity as determined by ESR spectroscopy in both oxidizing systems. Rosemary was an efficient radical scavenger in both systems, but behaved as a prooxidant on thiols in the OXHydro system. Acerola was also found to be prooxidative as determined by increased radical signal intensity especially in the OXLip system, assigned to high concentration of ascorbate in the extract. Natural nitrate sources, especially lettuce and spinach, are accordingly potential substitutes for synthetic phenolic antioxidants protecting against protein thiol oxidation and radical formation in pork.

Mate extract is superior to green tea extract in the protection against chicken meat protein thiol oxidation

Water soluble caffeic acid derivatives in aqueous extracts of mate (Ilex paraguariensis) showed a direct dose-dependent response, protecting protein thiols in a chicken breast meat model system when oxidation was initiated by a lipophilic radical initiator (2,2′-azobis(2,4-dimethylvaleronitrile; AMVN). In contrast, an inverse dose-dependent response was obtained when initiated by a hydrophilic radical initiator (2,2′-azobis(2-amidinopropane)dihydrochloride; AAPH), pointing toward a mechanism, where a specific concentration of water-soluble polyphenol yields optimum protection. Optimal concentrations of mate extract for the protection of thiols against radicals initiated in the lipid phase (AMVN) or in the aqueous phase (AAPH) were 0.1–0.5% and 0.01–0.1%, respectively. Green tea (Camellia sinensis) extract showed a pro-oxidative effect involving quinones from oxidation of pyrogallol-type catechins (epigallocatechin-3-gallate (EGCG), gallocatechin-3-gallate (GCG), and epicatechin-3-gallate (ECG)) and not the catechol-type catechins (catechin and epicatechin).

Impact of antioxidants dispersions on the stability and oxidation of water-in-olive-oil emulsions

Water-in-olive-oil emulsion stability was studied as a function of the composition of the water dispersed phase. In particular, different polyphenolic extracts from natural sources were dispersed in the olive oil and their impact on emulsion kinetic stability and susceptibility to oxidation was evaluated. As natural sources, extra virgin olive oil, olive mill waste and green tea leaves were chosen. To test their impact on emulsion properties, the emulsions were prepared with fixed aqueous phase content. As emulsifiers, a fixed percentage of a mixture Span 80 (sorbitan monoleate)/Tween 80 (polysorbate 80) was used. The effect of the antioxidant dispersion on emulsion oxidation was studied by triggering the oxidation reaction in the oil phase with the lipophilic radical initiator AMVN (2,2-azobis(2,4-dimethylvaleronitrile). Then, the oxidation reaction was followed by using diphenyl-1-pyrenylphosphine, which becomes fluorescent when it is oxidized by hydroperoxides. The impact of antioxidant dispersions on emulsion kinetic stability was studied by UV–Vis turbidity measurements. The oxidation results were correlated to antioxidant extracts oxygen radical adsorption capacity (ORAC) and to emulsion kinetic stability. On the whole, antioxidants dispersions delayed the oxidation reaction to different extents in dependence on their ORAC values and their components amphiphilicity. Remarkably, among the antioxidants tested, the aqueous polyphenol extract from virgin olive oil was the most effective because it protected emulsions both from oxidation and from phase separation. Additionally, from this set of experiments, the primary role of the interfacial properties of olive oil polyphenols was highlighted.

A fluorometric assay to determine antioxidant activity of both hydrophilic and lipophilic components in plant foods

This study aimed to develop a fluorometric method to determine total antioxidant activity of plant foods. The antioxidant activities in plant foods were determined after extracting (1) hydrophilic components with acidified methanol (methanol:glacial acetate acid:water=50:3.7:46.3), (2) lipophilic components with methanol followed by tetrahydrofuran (THF), or (3) both hydrophilic and lipophilic components using sequential extraction of acidified methanol and THF together. Both the hydrophilic assay [using the hydrophilic radical initiator 2,2′-azobis-(2-amidinopropane)dihydrochloride (10 mmol/L) and hydrophilic probe 2,7-dichlorodihydrofluorescein (DCFH)] and the lipophilic assay [using the lipophilic radical initiator [2,2′-azobis (4-methoxiy-2,4-dimethylvaleronitrile), 2 mmol/L], and the lipophilic probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene-3-undecanoic acid (C11-BODIPY 581/591) (BODIPY: 2 μmol/L)] were used to measure antioxidant activity. The inhibition of BODIPY oxidation was significantly increased ( P<.01) when both the hydrophilic and lipophilic components were extracted using acidified methanol and organic solvent as compared to those extracted by organic solvent alone. In addition, the rate of DCFH oxidation was significantly delayed ( P<.05) when both components coexisted compared to DCFH oxidation of the hydrophilic component alone. The combination of lipophilic and hydrophilic components in these plant foods showed significantly greater antioxidant activity than that of either hydrophilic or lipophilic component alone. Thus, both hydrophilic and lipophilic components in plant foods and their interactions should be considered when determining their antioxidant activity.

Interaction of wheat germ agglutinin with an N-acetylglucosamine-carrying telomer brush accumulated on a colloidal gold monolayer

A disulfide-carrying telomer with many pendent N-acetylglucosamine (GlcNAc) residues (Cys-PMHGlcNAc) was obtained by photo-polymerization of 1-(6′-methacryloylaminohexyl)-2- N-acetoamido-2-deoxy d-glucopyranoside) (MHGlcNAc) using a benzyl N, N-diethyldithiocarbamoyl (BDC) derivative that shows abilities of initiation, trans fer, and termination ( iniferter). The disulfide-carrying telomer was accumulated on a monolayer of colloidal Au on a glass substrate, and the interaction of wheat germ agglutinin (WGA) with GlcNAc residue at the polymer brush-solution interface was examined by using the localized surface plasmon resonance (LSPR) technique. For comparison, an amphiphile carrying many pendent GlcNAc residues was also prepared with MHGlcNAc and a lipophilic radical initiator and was incorporated in a phospholipid liposome to examine interaction of the GlcNAc residue with WGA on the liposome surface using turbidity measurements. Both the colloidal gold optical device and the liposome showed a concentration-dependent specific binding of WGA, and the GlcNAc-carrying liposome had a detection limit of 100 nM for WGA, whereas that of the colloidal gold device was 10 nM. The sugar-carrying telomer-coated device examined here is not only useful as a simple biosensor chip but is also expected to expand our knowledge of bio-related phenomena at the liquid-telomer brush interfaces on a colloidal Au.

Phytonutrients affecting hydrophilic and lipophilic antioxidant activities in fruits, vegetables and legumes

AbstractThe antioxidant activities of fruits (n = 21), vegetables (n = 67) and legumes (n = 7) commonly consumed in Korea were determined by both the lipophilic antioxidant performance assay (LAP) and the hydrophilic oxygen radical absorbance capacity assay (ORAC). The LAP assay used the lipophilic radical initiator MeO‐AMVN [2,2′‐azobis(4‐methoxy‐2,4‐dimethylvaleronitrile)] and the lipophilic probe BODIPY 581/591 [4,4‐difluoro‐5‐(4‐phenyl‐1,3‐butadienyl)‐4‐bora‐3a, 4a‐diaza‐s‐indacene‐3‐undecanoic acid]. The ORAC assay used the hydrophilic radical initiator AAPH [2,2′‐azobis(2‐amidinopropane) dihydrochloride] and the hydrophilic probe fluorescein. In addition, the lipid‐soluble phytonutrients, carotenoids and tocopherols were determined by a reverse‐phase HPLC system using a C30 column with a UV detector. The water‐soluble phytonutrient, ascorbic acid, was analyzed using an HPLC system with an electrochemical detector. Total phenols were determined by UV spectrophotometry. Tocopherols (r = 0.774, p &lt; 0.0001) and carotenoids (r = 0.569, p &lt; 0.0001) were significantly correlated with LAP in total samples (n = 95). ORAC was significantly correlated with total phenols (r = 0.893, p &lt; 0.0001), but not with ascorbic acid (r = 0.009, p = 0.929) in total samples (n = 95). These data indicate that carotenoids and tocopherols and total phenols are the major contributors to the lipophilic and hydrophilic antioxidant capacities, respectively. Therefore, the contribution of both the hydrophilic and lipophilic components of fruits and vegetables should be considered when determining the actual ‘total’ antioxidant activity of fruits and vegetables. Copyright © 2007 Society of Chemical Industry

Total antioxidant performance: A validated fluorescence assay for the measurement of plasma oxidizability

The antioxidant capacity of human plasma was determined by following the oxidation kinetics of the lipid-soluble fluorescent marker BODIPY using 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the lipophilic radical initiator. The results are expressed as a total antioxidant performance (TAP) value based on the inhibition of BODIPY oxidation, as determined by the appearance of green fluorescence, with respect to a control sample (phosphatidylcholine with or without delipidized human serum). The suitability of the assay was evaluated on the basis of its precision, reproducibility, and specificity. The intra- and interassay coefficients of variation both were less than 5%. The addition of a representative substrate of plasma peroxidation, phosphatidylcholine, up to 750 μg/ml did not induce significant changes in the TAP value. Also, BODIPY photooxidation was not observed during the experimental time course (220 min). The TAP values of 6 plasma samples from healthy donors were measured and correlated with the main plasma water- and lipid-soluble antioxidants (uric acid and ascorbic acid, α-tocopherol, and carotenoids) and lipid profiles. Significant correlations were found between TAP and uric acid ( R = 0.97, P < 0.05) and cholesterol-adjusted α-tocopherol ( R = 0.93, P < 0.01). The results confirm that the TAP assay is suitable to measure the antioxidant activity of plasma antioxidants localized in both the lipophilic and hydrophilic compartments.

(−)-Epigallocatechin-(3)-gallate prevents oxidative damage in both the aqueous and lipid compartments of human plasma

When human plasma was exposed to the hydrophilic radical initiator, AAPH, (−)-epigallocatechin-(3)-gallate (EGCG) dose-dependently inhibited the aqueous compartment oxidation ( IC 50=0.72 μM ) (monitored by DCFH oxidation) and spared the lipophilic antioxidants, α-tocopherol, and carotenoids, but not ascorbic acid. When radicals were selectively induced in the lipid compartment by the lipophilic radical initiator, MeO-AMVN, EGCG spared α-tocopherol, but not carotenoids and inhibited the lipid compartment oxidation (monitored by BODIPY 581/591) with a potency lower than that found in the aqueous compartment ( IC 50=4.37 μM ). Our results indicate that EGCG, mainly localized in the aqueous compartment, effectively quenches aqueous radical species, thus limiting their diffusion into the lipid compartment and preventing lipid-soluble antioxidant depletion. Further, ESR experiments confirmed that EGCG recycled α-tocopherol through a H-transfer mechanism at the aqueous/lipid interface affording an additional protective mechanism to the lipid compartment of plasma.

A lipophilic free radical initiator, 2,2′-azobis (2,4-dimethylvaleronitrile) (AMVN) enhances caspase-dependent apoptosis induced by hyperthermia

Purpose: A free radical initiator, 2,2'-azobis (2-amidinopropane) dehydrochloride (AAPH), was previously found to enhance apoptosis by hyperthermia. Here, but more lipophilic free radical initiator, 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) was investigated for its effects as a possible heat sensitizer. Materials and methods: Human myelogenous monocytic leukaemia U937 cells were treated with hyperthermia combined with a various concentration of AMVN for investigating its ability to induce apoptosis and various parameters to identify the pathway. Results: Combined treatment of hyperthermia and AMVN induced DNA fragmentation markedly, while hyperthermia or AMVN alone induced marginal DNA fragmentation. Fractions of cells showed low mitochondrial membrane potential and increased superoxide production after the combined treatment. Experiments using various caspase inhibitors and a fluorogenic monitor of caspase 3 activities indicated that caspase acts both up- and down-stream of mitochondria. Conclusions: AMVN is suggested to be a potential heat sensitizer effective at a lower concentration than AAPH. The possible mechanism is discussed.

A method to measure the oxidizability of both the aqueous and lipid compartments of plasma

The lipophilic radical initiator (MeO-AMVN) and the fluorescent probe C11BODIPY581/591 (BODIPY) were used to measure the lipid compartment oxidizability of human plasma. Aqueous plasma oxidizability was initiated by the aqueous peroxyl radical generator, AAPH, and 2′,7′-dichlorodihydrofluorescein (DCFH) was employed as the marker of the oxidative reaction. The distribution in aqueous and lipid compartments of the two radical initiators was determined by measuring the rate of consumption of the plasma hydrophilic and lipophilic endogenous antioxidants. In the presence of AAPH (20 mM), the order of consumption was: ascorbic acid > α-tocopherol > uric acid > β-carotene, indicating a gradient of peroxyl radicals from the aqueous to the lipid phase. When MeO-AMVN was used (2mM), β-carotene was consumed earlier than uric acid and almost at the same time as α-tocopherol, reflecting the diffusion and activation of MeO-AMVN in the lipophilic phase. The rate of BODIPY oxidation (increase in green fluorescence) significantly increased after the depletion of endogenous α-tocopherol and β-carotene, whereas it was delayed for 180 min when AAPH was used instead of MeO-AMVN. The measurement of lipid oxidation in plasma was validated by adding to plasma the two lipophilic antioxidants, α-tocopherol and β-carotene, whose inhibitory effects on BODIPY oxidation were dependent on the duration of the preincubation period and hence to their lipid diffusion. DCFH oxidation induced by AAPH only began after uric acid, the main hydrophilic plasma antioxidant, was consumed. In contrast, when MeO-AMVN was used, DCFH oxidation was delayed for 120 min, indicating its localization in the aqueous domain. In summary, the selective fluorescence method reported here is capable of distinguishing the lipophilic and hydrophilic components of the total antioxidant capacity of plasma.

Dehydration Effect on the Recognition of Amphiphiles with Many Pendent Mannose Residues by Concanavalin A†

Amphiphiles which carried many pendent mannose residues as side chains were prepared by telomerization of 2-methacryloyloxyethyl d-mannopyranoside (MEMan, α:β = 6.5:3.5) using a lipophilic radical initiator. The mannose-carrying amphiphiles (DODA−PMEMan, DP (degree of polymerization) = 3, 8, 14, and 17) incorporated in liposomes were recognized by a lectin from Canavalia ensiformis (Con A), which was proven by the increase in turbidity of the liposome suspension after mixing with the lectin. The recognition was largely affected by the degree of polymerization and surface density of the amphiphile in the liposomes. The association constant (Kas) between the mannose residue and Con A (2.2 × 105 M-1 for DODA-PMEMan (DP = 17) at 25 °C) was much larger than those for low molecular weight sugars (for example, methyl α-d-mannopyranoside, Kas = 8.2 × 103 M-1) due to the “cluster effect”. Thermodynamic parameters showed a significant contribution of positive entropy change to the effective sugar recognition in the liposome system, which was in contrast to the enthalpy-driven recognition of low molecular weight sugars by Con A. The positive entropy change could be attributed to the “dehydration” of both Con A molecule and the mannose-carrying polymer chains which were fixed to the gigantic and hydrophilic liposome surface.

Recognition of novel amphiphiles with many pendent mannose residues by Con A.

Novel amphiphiles which carry many mannose residues as side chains were prepared by telomerization of N-methacryloylaminopropyl D-mannopyranoside (alpha:beta = 20:1), N-methacryloylaminohexyl D-mannopyranoside (alpha:beta = 20:1), or 3-(2-methacryloylaminoethylthio)propyl D-mannopyranoside (alpha:beta = 4:1) using a lipophilic radical initiator. The mannose-carrying amphiphiles incorporated in liposomes were recognized by a lectin from Canavalia ensiformis (Con A), which was proven by the increase in turbidity of the liposome suspension after mixing with Con A. The interaction between sugar residues on the liposome surface and the lectin was largely affected by the degree of polymerization (DP) and the surface density of the amphiphile in the liposomes. The distance between the sugar residues and the polymer main chain did not affect the specific recognition by the lectin significantly in the liposome system, whereas it appreciably affected the recognition in the water-soluble polymer system. The association constants (Ka) of the amphiphiles (DP approximately 18) with Con A (0.3-2.2 x 10(6) M-1 at 25 degrees C) were much larger than that of alpha-methyl D-mannopyranoside (8.2 x 10(3) M-1) due to the "cluster effect ". The positive entropy change (20-52 J/mol K) for the binding of Con A to mannose residues on the liposome surface showed that the recognition in the liposome system was largely promoted by the release of water molecules from both the sugar residues on the liposome surface and the binding site of Con A.

Recognition of Amphiphiles with Many Pendent Galactose Residues byRicinus communisAgglutinin

Amphiphiles which carry many pendent galactose residues as side chains were prepared by telomerization of 2-methacryloyloxyethyl β-D-galactopyranoside (MEGal) or 3-(2-methacryloyl aminoethylthio)propylD-galactopyranoside (MEPGal, α:β = 3.9:1) using a lipophilic radical initiator. The galactose-carrying amphiphiles (DP (degree of polymerization) = 15) incorporated in liposomes were recognized by a lectin fromRicinus communis(RCA120), which was proven by the increase in turbidity of the liposome suspension after mixing with the lectin. The recognition was largely affected by the distance between the galactose residues and the polymer main chain, and the surface density of the amphiphile in the liposomes. The liposomes containing these galactolipids were not taken up by mouse peritoneal macrophages, probably due to a steric hindrance of polymer main chains from the uptake of D-galactose receptors on the macrophages.

薬用外でのリポソームの利用 機能性高分子を表層に有するリポソームの特性解析

Liposomes are frequently used as model systems to clarify diverse functions of biomembranes. We have been introducing various functional polymer chains on the surface of liposomes by using a lipophilic radical initiator. In this review, our recent studies concerning specific molecular recognition on liposome surfaces were introduced.

Catalytic properties of galactose oxidase to liposome-forming amphiphiles which have many pendent galactose residues.

A galactose-carrying vinyl monomer [2-(methacryloyloxy)ethyl beta-D-galactopyranoside, MEGal] was polymerized by using a lipophilic radical initiator. The amphiphiles obtained (DODA-PMEGal) formed stable liposomes by mixing with phospholipids, and the galactose residues on the liposome surface were effectively recognized and oxidized by galactose oxidase. The affinity (estimated by the 1/Km value) of galactose oxidase for the galactose residues on the liposomes was higher than those for free galactose and MEGal and dependent on the length of galactose-carrying polymer chains on the liposome surface and the fluidity of the membranes, whereas not significantly influenced by the surface density of galactose residues on the liposomes. The affinity of galactose oxidase for the galactose-carrying linear polymers, which were prepared by using an ordinary azo-type radical initiator and a chain-transfer reagent, was also higher than those for free galactose and MEGal and dependent on the degree of polymerization of MEGal. The affinity was, however, relatively much smaller than those for DODA-PMEGals incorporated in liposomes.

Interfacial Recognition of Sugars by Novel Boronic Acid-Carrying Amphiphiles Prepared with a Lipophilic Radical Initiator

Novel amphiphiles which carry several boronic acid groups in their polar head regions were prepared by copolymerization of 3-(acrylamido)phenylboronic acid (APBA) and ((N,N-dimethylamino)propyl)methacrylamide (DMAPMA) using a lipophilic radical initiator. The amphiphiles obtained (Lipid AD) formed a stable monolayer, and the surface pressure−molecular area (π−A) profile of the monolayer was changed by the addition of sugars in the subphase. The effect was dependent on the structure of the sugars added, which is consistent with the effect of the sugars on the viscosity of aqueous solution of the copolymer of APBA and DMAPMA. The presence of DMAPMA residues together with the APBA residues in the head group of the amphiphile was more effective on the recognition of sugars than that of acrylamide or (3-(methacryloylamino)propyl)trimethylammonium chloride residues, which confirmed the supporting effect of tertiary amines on the recognition of sugars by phenylboronic acid groups previously reported. The monolay...

Pyrene-labeled amphiphilic poly-(N-isopropylacrylamides) prepared by using a lipophilic radical initiator: synthesis, solution properties in water, and interactions with liposomes

Fluorescently labeled amphiphilic poly-(N-isopropylacrylamides) (PNIPAM) substituted with a N-[4-(1-pyrenyl)butyl]-N-n-octadecyl group at the chain end were prepared by free-radical polymerization in dioxane of N-isopropylacrylamide (NIPAM) using 4,4′-azobis{4-cyano-N,N-[4-(1-pyrenyl)butyl]-n-octadecyl}pentanamide as the initiator. The solution properties of the polymers in water were studied as a function of polymer concentration and temperature. Quasi-elastic light-scattering measurements and fluorescence experiments monitoring the pyrene excimer and pyrene monomer emissions revealed the presence of multimolecular polymeric micelles below the lower critical solution temperature (LCST) of PNIPAM. These underwent partial, reversible reorganization as they were heated above the LCST. The interactions of the pyrene-labeled amphiphilic PNIPAM with dimyristoylphosphatidylcholine (DMPC) liposomes have been examined in water at 25 °C. From fluorescence experiments it was established that the polymeric micelles are disrupted irreversibly upon contact with the liposomes. The anchoring of the polymer chains occurs by insertion of their hydrophobic tail within the phospholipidic bilayer, as evidenced from a large decrease of the pyrene excimer emission relative to pyrene monomer emission. The copolymers remained anchored within the bilayer as the temperature of the copolymer–liposome suspension was raised above the LCST of PNIPAM. Keywords: liposome, poly-(N-isopropylacrylamide), fluorescence, micelles.

Galactose-containing amphiphiles prepared with a lipophilic radical initiator: association processes between liposomes triggered by enzymatic reaction.

A galactose-containing monomer (2-(methacryloyloxy)ethyl beta-D-galactopyranoside, MEGal) was polymerized by using a lipophilic radical initiator. The amphiphile obtained formed a liposome by mixing with bis(trans,trans-2,4-dioctadecadienoyl)phosphatidylcholine (DDPC), and the liposome obtained was physically stabilized by the polymerization of DDPC by UV irradiation. The enzymatic treatment of the galactose-containing liposomes with galactose oxidase resulted in the formation of aldehyde groups on the liposome surface. By the subsequent mixing of the liposome suspension with the amino group-containing liposome suspension, a rapid increase in turbidity was observed due to the formation of Schiff bases between the aldehyde groups and the amino groups at the interface of the liposomes. The rate of turbidity change strongly depended on the degree of polymerization of MEGal, the surface densities of galactose and amino groups on the liposome, the distance from the liposome surface to amino end groups, and the flexibility and deformability of the liposomes.

Galactose-containing amphiphiles prepared with a lipophilic radical initiator.

Novel amphiphiles which contain galactose residues (degree of polymerization (DP) = 6.2, 10, and 15) were prepared by telomerization of 2-[(methacryloyloxy)ethyl]-beta-D-galactopyranoside using a lipophilic radical initiator. The galactose-carrying amphiphile incorporated in a liposome was recognized by a lectin from Ricinus communis (RCA120), which was proven by the increase in turbidity of the liposome suspension after mixing with the lectin. The recognition was largely affected by the degree of polymerization and the surface density of the amphiphile. The amphiphile would be useful as a component of the drug delivery system to hepatocytes.

Sugar-Containing Lipids Prepared by Using a Lipophilic Radical Initiator: Interfacial Recognition by Lectin As Studied by Using the Multiple Internal Reflection Fluorescence Method

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSugar-Containing Lipids Prepared by Using a Lipophilic Radical Initiator: Interfacial Recognition by Lectin As Studied by Using the Multiple Internal Reflection Fluorescence MethodHiromi Kitano and Kohji OhnoCite this: Langmuir 1994, 10, 11, 4131–4135Publication Date (Print):November 1, 1994Publication History Published online1 May 2002Published inissue 1 November 1994https://doi.org/10.1021/la00023a036RIGHTS & PERMISSIONSArticle Views106Altmetric-Citations31LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (548 KB) Get e-Alerts Get e-Alerts