Acids In Barley Research Articles

Heterologous expression and enzymatic characteristics of sulfatase from Lactobacillus plantarum dy-1.

Barley, rich in bioactive components including dietary fiber, polyphenolic compounds and functional proteins, exhibits health benefits such as regulating glucose and lipid metabolism. Previous studies have found that the content and composition of free phenolic acids in barley may be significantly changed by fermentation with the laboratory patented strain Lactobacillus plantarum dy-1 (L. p dy-1), but the mechanism of enzymatic release of phenolic acid remains to be elucidated. Based on this, this study aimed to identify the key enzyme in L. p dy-1 responsible for releasing the bound phenolic acid and to further analyze its enzymatic properties. The Carbohydrate-Active enZYmes database revealed that L. p dy-1 encodes 7 types of auxiliary enzymes, among which we have identified a membrane sulfatase. The enzyme gene LPMS05445 was heterologous to that expressed in E. coli, and a recombinant strain was induced to produce the target protein and purified. The molecular weight of the purified enzyme was about 59.9 kDa, with 578.21 U mg-1 enzyme activity. The optimal temperature and pH for LPMS05445 expression were 40 °C and 7.0, respectively. Furthermore, enzymatic hydrolysis by LPMS05445 can obviously change the surface microstructure of dietary fiber from barley bran and enhance the release of bound phenolic acid, thereby increasing the free phenolic acid content and improving its physiological function. In conclusion, sulfatase produced by Lactobacillus plantarum dy-1 plays a key role in releasing bound phenolic acids during the fermentation of barley.

Determination and prediction of digestible energy, metabolizable energy, and standardized ileal digestibility of amino acids in barley for growing pig

Two experiments were performed to determine the digestible energy (DE), metabolizable energy (ME) and standardized ileal digestibility coefficients (SIDC) of amino acids (AA) in barley for pigs, and to develop prediction equations for the DE, ME and SIDC of AA based on its chemical composition. In Exp. 1, twenty-two castrated male pigs (29.5 ± 1.3 kg) were allotted to a replicated 11 × 3 incomplete Latin square design, including 11 diets and 3 consecutive periods. A corn-soybean meal basal diet and 10 test diets containing 400 g/kg of each barley at the expense of corn and soybean meal were prepared, and all diets had a constant ratio of corn to soybean meal. Each period includes 7 d of adaption and 5 d of total feces and urine collection. The DE and ME contents were measured by the total collection method. In Exp. 2, twenty-two ileal-cannulated pigs (30.5 ± 2.1 kg) were used in a duplicated 11 × 3 incomplete Latin square design with 11 diets and three 7-day periods. Titanium dioxide (3 g/kg) was added to all diets as indicator and ieal digesta were collected on d 6 and 7 of each period. Barley was the sole nitrogen source in 10 barley diets and a nitrogen-free diet was used for the endogenous nitrogen losses. The results showed great variations in ether extract (EE), calcium (Ca), ash, acid detergent fiber (ADF) and crude fiber (CF) among samples of barley. On an air-dry basis, the DE and ME in barley for pigs were 12.26 (11.29–13.58) MJ/kg and 11.87 (10.95–13.15) MJ/kg, respectively. The optimal prediction equations for DE and ME were: DE (MJ/kg) = −23.594 + 0.358 crude protein (CP) + 0.043 EE (R2 =0.95) and ME (MJ/kg) = 0.338 + DE - 0.014 ADF (R2 =0.99), respectively. The SIDC of lysine (Lys), methionine (Met), threonine (Thr), and tryptophan (Trp) in barley were 0.767 (0.731–0.809) g/kg, 0.825 (0.803–0.872) g/kg, 0.809 (0.793–0.822) g/kg, and 0.794 (0.760–0.858) g/kg, respectively. The best-fit prediction equations for SIDC of Lys, Met, Thr and Trp in barley fed to pigs were: SIDC Lys = 0.721 + 0.002 EE (R2 =0.79), SIDC Met = 1.045–0.00041 total starch (R2 =0.69), SIDC Thr = 0.739 + 0.001 ADF (R2 =0.69) and SIDC Trp = 1.468–0.006 CP - 0.001 neutral detergent fiber (R2 =0.99), respectively. The study confirms the large variations in chemical composition of different sources of barley, which contributes to the differences of DE, ME and SIDC of AA in barley fed to pigs and the fiber fraction can be used as key predictors.

Mechanism of calcium in melatonin enhancement of functional substance-phenolic acid in germinated hulless barley.

Phenolic acid is a physiologically active substance that has a variety of effects on humans. Barley sprouts are often used as food ingredients to enrich phenolic acids and to further produce functional foods rich in phenolic acids. In this study, the mechanism of Ca2+ involvement in regulating phenolic acid biosynthesis and plant growth in barley by melatonin (MT) under NaCl stress was investigated. According to the studies, MT (25 μM) increased total calcium content, induced Ca2+ burst, and up-regulated the gene expression of calcium-regulated protein-dependent protein kinase and calcium-binding protein transcription-activating protease in NaCl-stressed (60 mM) barley. Exogenous MT and its combined CaCl2 (0.4 mM) significantly promoted phenolic acid biosynthesis by increasing the activity of C4H and PAL, and induced gene expression of PAL and F5H. The addition of exogenous CaCl2 and MT caused systemic tolerance in NaCl-stressed barley, as determined by a decrease in the fluorescence intensity of hydrogen peroxide and oxygen radical anions as well as an enhancement in the antioxidant enzyme, thus significantly increasing sprout length and fresh weight. In addition, combined use of MT with Ca2+ antagonists (lanthanum chloride or ethylene glycol tetraacetic acid), impaired all impacts as mentioned above. These findings imply that Ca2+ participated in MT-induced phenolic acid biosynthesis and growth improvement in NaCl-stressed barley.

Effect of grinding and extraction conditions on the determination of antioxidant activity and phenolic acids in barley

In the present study, the objective was to evaluate the effects of barley kernel size, grind level, and extract solvent on the antioxidant activities associated with total phenolic content and phenolic acid compositions. Three barley varieties (Kindred, Azure, and Tradition), were used and the results showed that with the exception of superoxide anion radical scavenging activity, thin kernel size fraction (2.0 mm) showed much higher antioxidant activities and total phenolic content than those in the 2.4 mm and 2.8 mm fractions when averaged across barley cultivar, grind level, and extract solvent. A similar trend was found for individual phenolic acid compositions among kernel sizes. Simple correlation analysis revealed that total phenolic content showed strong correlation with DPPH· scavenging activity, ABTS + scavenging activity, and reducing power. Generally, fine grind and 80% acetone extract showed averaged highest values in antioxidant activities, total phenolic content, and individual phenolic acid compositions. Stepwise linear regression showed that extract solvent was the most important factor for DPPH· scavenging activity, ABTS + scavenging activity, reducing power, and iron chelating activity. The sum of syringic acid and caffeic acid exhibited a dominant role in explaining the major variation in antioxidant activities except for superoxide anion radical scavenging activity.

Identification of QTLs associated with p-coumaric acid and ferulic acid in barley

Phenolic acids in plants is receiving more attention because of their nutritive function and essential roles in plant growth. However, the genetics of phenolic acids in barley is still unclear. In this study, two main phenolic acids, i.e. ferulic acid (FA) and p-coumaric acid (p-CA), were analyzed in the grains of a double haploid population derived from XZ153 (Tibetan wild barley) × Hua30 (cultivated cultivar) grown in three locations. A wide variation in the content of phenolic acids was observed among the DH population. Quantitative trait locus (QTL) analysis of phenolic acid content in barley grains from three locations totally identified 4 QTLs associated with p-CA content and 4 QTLs with FA content. Based on the mean phenolic acids content over the three locations, two significant QTLs (M_3261686 for p-CA and M_4184755 for FA), belonging to cytochrome P450 family (HvCYPs) were identified, with the LOD value of 5.33 and 7.01 respectively. These two QTLs could account for 14% of phenotypic variation for FA and 18% for p-CA. Collinearity analysis revealed that HvCYPPA (QTL for p-CA) has duplication events in barley genome. Furthermore, there was no synteny block found for these two genes in barley genome. The current results provide a new knowledge about genetic controlling of phenolic acids synthesis in barley grains.

Fatty acids, sterols and tocols in Irish barley varieties: Profiling and correlation analysis

The objective of this study was determination and comparison of three types of lipophilic phytochemicals, fatty acids, phytosterols and tocols, in five wholegrain Irish barley varieties. Fatty acids and sterols were derivatised and analysed by gas chromatography, while tocols were analysed by high performance liquid chromatography. The content of saturated and unsaturated fatty acids in the varieties ranged between 442–2615 and 1200–1730 mg/100 g dw, respectively, indicating abundance of unsaturated fatty acids in barley. Linoleic acid was the most abundant unsaturated fatty acid. Phytosterols in the barley varieties ranged between 66 and 82 mg/100 g dw with β‐sitosterol being the most abundant sterol, while tocols ranged between 46 and 58 µg/g dw with α‐tocotrienol being the most abundant tocol homologue. Principle component analysis (PCA) revealed interesting correlations between these phytochemicals. An evident relationship between the unsaturated fatty acids and some tocol homologues was observed. Sterols like β‐sitosterol and β‐sitostanol were negatively correlated with each other. PCA also indicated possible genotypic relationships between the barley varieties.Practical applications: Barley lipids serve as a considerable source of major fatty acids, phytosterols and tocols. By means of fractionation such as milling, enriched sources of these phytochemicals for use as ingredients in functional foods might be produced. Concurrent analysis of phytochemicals undertaken in this study, along with determination of correlations between them provides useful information to geneticists and plant breeders, in choosing specific varieties/genotypes and producing crossbreeds with improved levels of certain phytochemicals.Analysis of fatty acids, sterols and tocols in barley using chromatographic techniques, and determination of correlations between them by principal component analysis (PCA).

Determination of basal ileal endogenous losses and standardized ileal digestibility of amino acids in barley fed to growing pigs.

BackgroundBasal ileal endogenous amino acid (AA) losses (IAAend) and standardized ileal digestibility (SID) values of cereal grains, such as barley, are apparently underestimated when determined according to the nitrogen (N)-free method. Regression analysis between the dietary apparent ileal digestible content (cAID) and total crude protein (CP) and AA can be considered as alternative approach to obtain more accurate values for IAAend and SID of AA in cereal grains.MethodsEight hulled barley genotypes were used, with barley being the only source of CP and AA in the assay diets. The diets contained 95 % as-fed of these eight barley genotypes each, ranging in CP content between 109.1 and 123.8 g/kg dry matter (DM). Nine ileally T-cannulated barrows, average body weight (BW) 30 ± 2 kg, were allotted to a row-column design comprising eight periods with 6 d each and nine pigs. On d 5 and the night of d 6 of every period, ileal digesta were collected for a total of 12 h. The IAAend and the SID were determined by linear regression analysis between cAID and total dietary CP and AA.ResultsThere exist linear relationships between cAID and total CP and AA (P < 0.001). The IAAend of CP, Lys, Met, Thr and Trp amounted to 35.34, 1.08, 0.25, 1.02 and 0.38 g/kg DM intake (DMI), respectively, which are greater compared to average IAAend determined previously under N-free feeding conditions. The SID of CP, Lys, Met, Thr and Trp was 90, 79, 85, 79 and 86 %, respectively, and was greater when compared to tabulated values. Moreover, these SID values were greater than those reported in literature, based on correction of apparent ileal digestibility (AID) of CP and AA for their IAAend values. Summarized, the results of the present regression analysis indicate greater IAAend in barley-based diets compared to those obtained by N-free feeding.ConclusionsFor low-protein feed ingredients like barley the regression method may be preferred over correction of AID values for their IAAend determined under N-free feeding conditions, as intercepts and slopes of the linear regression equations between cAID and total dietary CP and AA provide direct estimates of IAAend and SID of CP and AA in the presence of the assay feed ingredient.

Genetic Diversity of Individual Phenolic Acids in Barley and Their Correlation with Barley Malt Quality.

Phenolic acids have been quite extensively studied in food science research because of their antioxidative effect. In this study, the genotypic difference and genetic control of phenolic acids, and their correlation with malt quality, were investigated in barley. Ferulic acid (FA) and p-coumaric acid (p-CA) were identified as two main phenolic acids, showing wide variations among 68 barley genotypes. The mean content of FA and p-CA were 2.15 μg g(-1) and 1.10 μg g(-1) in grains and 4.07 μg g(-1) and 1.44 μg g(-1) in malt, respectively. After malting, FA and p-CA were increased significantly in 55 and 37 genotypes and were reduced in 2 and 14 genotypes, respectively. Both malt FA and p-CA were positively correlated with soluble N content and Kolbach index and negatively correlated with malt extract and viscosity. The results indicated that the effect of malting on the change of an individual phenolic acid is genotype independent. Association mapping identified that 8 markers on Chromosomes 1H, 2H, 4H, and 7H are associated with grain p-CA and 4 markers on Chromosomes 3H and 7H are linked with grain FA. However, only a single marker on Chromosome 3H was found to be associated with malt FA. Moreover, a lack of overlapping markers between grain and malt indicated the genetic diversity of phenolic acids in barley grain and malt. Our results strengthen the understanding of phenolic acids in barley and their responses to the malting process.

Apparent ileal digestibility of amino acids and metabolisable energy value in grains for broilers

Using ileal digestible amino acids in feed optimising will intensify feed protein utilizing and decrease nitrogen excretion to the environment. The study determined the apparent ileal digestibility (AID) coefficients of amino acids in barley, wheat, oats, triticale, maize, and dehulled oats in the diets of 180 Ross broiler chickens (aged 24–35 days). The birds were fed semi-purified diets that contained grain as the sole protein source and chromium-mordanted straw as an indigestible marker. The AID coefficients of the nutrients were assessed using the slaughter technique, and the apparent metabolisable energy (AME) was determined using total excreta collection. The ileal digestibility of the dry matter and organic matter were the highest in maize. The AME of maize was higher than that of other cereals. The ileal digestibility of crude protein was higher in wheat than that in barley, oats and dehulled oats. The AME of wheat was similar to that of barley and oats but lower than that of triticale and dehulled oats. The amino acid AID was highest in wheat (0.86) and triticale (0.85) and lowest in oats (0.79) and barley 0.77). The average amino acid AID was 0.81 in dehulled oats. The threonine AID was the same in all tested ingredients. The lysine, methionine, and cystine AID coefficients were 0.81, 0.79, and 0.71 respectively for barley; 0.86, 0.84, and 0.38 respectively for oats; 0.87, 0.86, and 0.53 respectively for dehulled oats; 0.84, 0.90, and 0.66 respectively for maize; 0.89, 0.88, and 0.77 respectively for triticale; and 0.87, 0.85, and 0.71 respectively for wheat. Results indicated that AME –values of domestic grains (barley, oats and wheat) are in the same level. Especially, low AME value of wheat needs further investigation.;

Additivity and Associative Effects of Metabolizable Energy and Amino Acid Digestibility in Barley and Canola Meal for White Pekin Ducks

An experiment was conducted using the TMEn bioassay method to investigate the additivity and associative effects of metabolizable energy and amino acid digestibility in barley and canola meal for White Pekin ducks. Additivity was tested by comparing the difference between observed values determined in a complete diet and predicted values from measurements determined with individual ingredients (barley and canola meal). Six ducks each were assigned to diets of barley, canola meal, the complete diet, and dextrose. Dextrose-fed ducks were used for estimation of endogenous losses for calculation of true amino acid digestibility. The observed AME, TME, AMEn, and TMEn values in the complete diet were 0.065, 0.083, 0.016, and 0.023 (kcal/g), respectively, numerically higher than predicted values. Differences between observed and predicted values were not significant (P > 0.05), indicating that the AME, AMEn, TME, and TMEn in barley and canola meal were all additive. In general, observed values for apparent amino acid digestibility (AAAD) and true amino acid digestibility (TAAD) in the complete diet were higher than those predicted from individual ingredients. Observed AAAD for lysine, histidine, tryptophan, alanine, and aspartate were higher (P < 0.05) than predicted values, indicating that digestibilities of these amino acids were not additive. The mean of AAAD in canola meal (77.29%) was higher (P < 0.05) than the observed values of barley (52.2%) and complete diet (64.55%). For TAAD values, differences between observed and predicted values were significant for lysine, histidine, and tryptophan (P < 0.05). The mean of TAAD in canola meal, barley, and complete diet were 85.88, 80.87, and 81.33%, respectively. The average difference between observed and predicted values for TAAD (1.18 %) was smaller than that of AAAD (5.41%). These results indicated that ME values for barley and canola meal were additive in the complete diet but that digestibilities of some amino acids were not additive; they further suggested that there were some associative effects of amino acids in barley and canola meal for ducks.

Ruminal degradability and intestinal digestibility of protein and amino acids in barley and oats expander-treated at various intensities

The objectives of the experiment were to study effects of pelleting or expander treatment at various intensities (mild, medium, hard or maximum) prior to pelleting at two temperatures in the mixer-conditioner (60 and 75°C) on ruminal degradation and intestinal digestibility of protein and amino acids in barley and oats, using nylon bag methods. Temperatures achieved were 85–95, 100–110 and 115–125°C at mild, medium and hard intensity, whereas maximum temperature achieved was 125°C for barley and 140°C for oats. Thus, temperatures higher than 130–135°C appeared unrealistic under commercial conditions. Pelleting decreased ruminal degradation of protein, especially at high temperature in the mixer-conditioner. Expander treatment decreased ruminal degradation of protein further and the lowest effective protein degradability (EPD) achieved was 30% for barley and 29% for oats, both obtained at maximum temperature in the expander and high temperature in the mixer-conditioner. These values are low compared to previous results and need to be verified. No negative effects of treatment on digestibility of protein were observed. This indicates that the treatments shifted site of protein digestion from the rumen to the small intestine. Expander treatment did not alter the contents of any individual amino acid either in barley or in oats. Expander treatment reduced ruminal degradation of total amino acids to the same extent as protein. The variation in ruminal degradation among amino acids was considerable. Thus, ruminal degradation of protein cannot be used for determination of ruminal degradation of all individual amino acids. Expander treatment did not increase the content of indigestible amino acids in barley or oats, indicating that amino acids were not heat-damaged.

Determination of apparent ileal amino acid digestibility in barley and canola meal for pigs with the direct, difference, and regression methods1

Three methods were evaluated for the determination of apparent ileal digestibility values of amino acids in feedstuffs with a low protein (barley, 10.2% CP) and a high protein content (canola meal, 38.2% CP). Five barrows, average initial BW 40 kg, were fitted with a simple T-cannula at the distal ileum and fed five diets according to a 5 x 5 Latin square design. Diet 1 contained 42.7% canola meal providing the sole source of dietary amino acids. Diets 2, 3, and 4 contained three graded levels of barley (22.5, 45.0, and 67.5%, respectively) and three graded levels of canola meal (36.6, 30.5, and 24.4%, respectively). Diet 5 contained 90.0% barley, which provided the sole source of dietary amino acids. With the exception of diet 5, the diets were formulated to contain 16% CP. Chromic oxide (.4%) was included as the digestibility marker. The pigs were fed twice daily, equal amounts, at 0800 and 2000. The dietary allowance was 1,800 g/d. Each experimental period comprised 8 d. Ileal digesta were collected for a total of 24 h during d 7 and 8 at 2-h intervals. Apparent ileal digestibility values of amino acids in barley were determined with the direct method from diet 5, with the difference method from diets 2, 3, and 4, and with the regression method from diets 1, 2, 3, and 4. Digestibility values of amino acids in canola meal were determined with the direct method from diet 1, with the difference method from diets 2, 3, and 4, and with the regression method from diets 1, 2, 3, and 4. There were no differences (P < .05) in the digestibility values in barley between the difference method when barley was included at 67.5% in the diet and the regression method. However, the digestibility values were lower (P < .05 or < .10) when these were determined with the direct method. There were no differences (P > .05) in the digestibility values of canola meal when these were determined with the direct method, the difference method, when canola meal was included at 36.6% in the diet, and the regression method. In conclusion, amino acid digestibility values in feedstuffs with a low protein content should be determined with the difference or regression methods rather than with the direct method. Amino acid digestibility values in feedstuffs with a high protein content can be determined with either method.

Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts

Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using (14)C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.

Physico‐chemical factors affecting uptake by roots and translocation to shoots of weak acids in barley

AbstractThe uptake from solution of maleic hydrazide, flamprop and a series of phenoxyacetic acids by roots, and their subsequent translocation to shoots, was measured in barley. Both uptake and translocation increased as the pH of the solution decreased, the magnitude of the change varying amongst the chemicals tested. Uptake by roots could be accounted for by the ion‐trap mechanism, which assumes that entry of the chemicals occurs largely by passive diffusion of the undissociated form of the acids, with passage of the anions across the cell membranes being very slow. The ratio of the permeability of the cell membranes to the undissociated and dissociated forms of the acids was estimated from the accumulation in roots, and in the phenoxyacetic acid series this ratio was maximal (4×105) for compounds of intermediate lipophilicity. Maleic hydrazide and flamprop had much lower ratios, 1.8×102 and 103 respectively; the value for flamprop was much less than for phenoxyacetic acids of similar lipophilicity, such as 2, 4‐ dichlorophenoxyacetic acid, indicating that lipophilicity may not be the sole factor determining the behaviour of weak acids in plants. Translocation to shoots was approximately proportional to the chemical concentrations in the roots.

Distribution of gramine and hydroxamic acids in barley and wheat leaves

The first leaf of 12-day-old barley seedlings, contained 1.3 μmol/g fr. wt gramine while the concentrations were 2.2, 2.0 and 3.1 μmol/g fr. wt in lower and upper epidermis and mesophyll parenchyma, respectively. Gramine was not detected in the vascular bundles, nor in xylem exudates or guttation drops. Thus, about 70% of total gramine in the leaf was found in the mesophyll parenchyma and 30% in epidermal tissue. The content of hydroxamic acids was 3.0 μmol/g fr. wt in the first leaf of 12-day-old wheat plants, while the concentrations were 6.8 and 4.1 μmol/g fresh wt in the vascular bundle and mesophyll parenchyma, respectively. These compounds were not detected in epidermis, xylem exudates and guttation drops. About 50% of total hydroxamic acids were found in the vascular bundles. The significance of these results in plant protection against aphids is discussed.

The Effect of Red and Far Red Light on the Desaturation of Fatty Acids in Barley Leaves

Barley (Hordeum vulgare) leaf tissue was either (a) exposed to continuous red light or (b) exposed to red, far red, or red followed by far red light. The fatty acid composition and incorporation of acetate-2-(14)C into linolenate were determined. Changes occurred in the fatty acid composition of dark-grown barley leaves regardless of whether the plants were subsequently exposed to red light or whether the tissue remained in the dark. Measurements were also made of the fatty acids of the coleoptile. Red light treatment did not reduce the lag period for the synthesis of linolenate when chlorophyll synthesis was inhibited. It appears that the desaturation process per se in the synthesis of linolenate is not phytochrome-mediated but may appear to be phytochrome mediated if, possibly, galactolipid and chlorophyll syntheses occur concomitantly.

SOME ORGANIC ACIDS IN BARLEY, MAIZE, OATS AND RYE PLANTS

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSOME ORGANIC ACIDS IN BARLEY, MAIZE, OATS AND RYE PLANTSE. K. Nelson and H. H. MotternCite this: J. Am. Chem. Soc. 1931, 53, 8, 3046–3048Publication Date (Print):August 1, 1931Publication History Published online1 May 2002Published inissue 1 August 1931https://doi.org/10.1021/ja01359a028RIGHTS & PERMISSIONSArticle Views61Altmetric-Citations18LEARN 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 (221 KB) Get e-Alerts Get e-Alerts