Inhibition of Xanthine Oxidase Activity by Gnaphalium Affine Extract

Objective To evaluate the inhibitory effect of Gnaphalium affine extracts on xanthine oxidase(XO) activity in vitro and to analyze the mechanism of this effect. Methods In this in vitro study, Kinetic measurements were performed in 4 different inhibitor concentrations and 5 different xanthine concentrations(60, 100, 200, 300, 400 μmol/L). Dixon and Lineweaver-Burk plot analysis were used to determine Ki values and the inhibition mode for the compounds isolated from Gnaphalium affine extract. Results Four potent xanthine oxidase inhibitors were found in 95% ethanolic(v/v) Gnaphalium affine extract. Among them, the f lavone Eupatilin exhibited the strongest inhibitory effect on XO with a inhibition constant(Ki) of 0.37 μmol/L, lower than the Ki of allopurinol(4.56 mol/L), a known synthetic XO inhibitor. Apigenin(Ki of 0.56 μmol/L, a proportion of 0.0053‰ in Gnaphalium affine), luteolin(Ki of 2.63 μmol/L, 0.0032‰ in Gnaphalium affine) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone(Ki of 3.15 μmol/L, 0.0043‰ in Gnaphalium affine) also contributed to the inhibitory effect of Gnaphalium affine extract on XO activity. Conclusions These results suggest that the use of Gnaphalium affine in the treatment of gout could be attributed to its inhibitory effect on XO. This study provides a rational basis for the traditional use of Gnaphalium affine against gout.

Xanthine oxidase inhibitors from Archidendron clypearia(Jack.) I.C. Nielsen: Results from systematic screening of Vietnamese medicinal plants

Objective: To screen Vietnamese medicinal plants for xanthine oxidase(XO) inhibitory activity and to isolate XO inhibitor(s) from the most active plant. Methods: The plants materials were extracted by methanol. The active plant materials were fractionated using different organic solvents, including n-hexane, ethyl acetate, and n-butanol. Bioassay-guided fractionation and column chromatography were used to isolate compounds. The compounds structures were elucidated by analysis of spectroscopic data, including IR, MS, and NMR. Results: Three hundreds and eleven methanol extracts(CME) belonging to 301 Vietnamese herbs were screened for XO inhibitory activity. Among these plants, 57 extracts displayed XO inhibitory activity at 100 μg/m L with inhibition rates of over 50%. The extracts of Archidendron clypearia, Smilax poilanei, Linociera ramiflora and Passiflora foetida exhibited the greatest potency with IC_(50) values below 30 μg/m L. Chemical study performed on the extract of Archidendron clypearia resulted in the isolation of six compounds, including 1-octacosanol, docosenoic acid, daucosterol, methyl gallate, quercitrin and(-)-7-O-galloyltricetiflavan. The compound(-)-7-O-galloyltricetiflavan showed the most potent XO inhibitory activity with an IC_(50) value of 25.5 μmol/L. Conclusions: From this investigation, four Vietnamese medicinal plants were identified to have XO inhibitory effects with IC_(50) values of the methanol extracts below 30 μg/m L. Compound(-)-7-O-galloyltricetiflavan was identified as an XO inhibitor from Archidendron clypearia with IC_(50) value of 25.5 μmol/L.

Identifi cation of egg protein-derived peptides as xanthine oxidase inhibitors:virtual hydrolysis,molecular docking,and in vitro activity evaluation

The purpose of this study was to screen the xanthine oxidase(XO)inhibitory peptides from egg white proteins through virtual hydrolysis,in vitro activity validation,and molecular docking.The results demonstrated that tripeptide EEK from ovalbumin exhibited potent XO inhibitory activity with an IC50 value of 141μmol/L.The molecular docking results showed that tripeptide EEK bound with the active center of XO via 3 carbon hydrogen bond interactions,2 salt bridges,5 conventional hydrogen bond interactions,and 4 attractive charge interactions.The residues Glu802,Phe1009,and Arg880 may play key roles in the XO catalytic reaction.Especially,the key intermolecular forces of inhibiting XO activity may be special type of hydrogen bonds including carbon hydrogen bond interactions and attraction charge interactions.The novel tripeptide EEK is potential candidates for controlling hyperuricemia.

Study of Xanthine Oxidase Activity in Sera of Iraqi Children with Nephrotic Syndrome

Introduction: To investigate the relationship of xanthine oxidase activity with nephrotic syndrome disorder in children, and the optimization of the enzyme activity conditions in the disorder. Material and methods: Sera of children with nephrotic syndrome (NS) (60 samples) were obtained from Central Child Teaching Al Karama Hospital (CCTAH), from 2nd Mar. 2013 to 28th Feb. 2014. Sera of the patients were assayed for xanthine oxidase (XO) activity using colorimetric absorbance technique. The obtained results were compared with the enzyme activity of normal children (70 samples) as control. Results and conclusions: The results revealed a significant (P < 0.001) elevation in XO activity in serum of nephrotic syndrome (0.12 ± 0.06 IU/L) compared with that of normal subjects (0.05 ± 0.009 IU/L), showing an elevation of (70%) in XO activity (about 2/3) that of normal group. Factors influencing XO activities were also studied and showed that XO activity is a pH dependent. Significant elevation (P < 0.001) was found in uric acid level in sera of NS patients (497.52 ± 3.21 μmol/L) compared with that in normal group (298.12 ± 1.70 μmol/L). Elevation was found in urea level in sera of NS patient (10.69 ± 7.55 mmol/L) compared with that of normal group (4.57 ± 1.27 mmol/L). It was appeared, there is a role of XO activity in the pathogenesis of endothelial injury during glomerular lesion in NS and that was confirmed by comparing XO activity and other related conditions with the activity of normal volunteers.

Determination of inhibitory activity of Salvia miltiorrhiza extracts on xanthine oxidase with a paper-based analytical device

A novel paper-based analytical device(PAD)was prepared and applied to determine the xanthine oxidase(XOD)inhibitory activity of Salvia miltiorrhiza extracts(SME).First,polycaprolactone was 3D printed on filter paper and heated to form hydrophobic barriers.Then the modified paper was cut according to the specific design.Necessary reagents including XOD for the colorimetric assay were immobilized on two separate pieces of paper.By simply adding phosphate buffer,the reaction was performed on the double-layer PAD.Quantitative results were obtained by analyzing the color intensity with the specialized device system(consisting of a smartphone,a detection box and sandwich plates).The 3Dprinted detection box was small,with a size of 9.0 cm×7.0 cm×11.5 cm.Color component G performed well in terms of linearity and detection limits and thus was identified as the index.The reaction conditions were optimized using a definitive screening design.Moreover,a 10%glycerol solution was found to be a suitable stabilizer.When the stabilizer was added,the activity of XOD could be maintained for at least 15 days under 4℃ or-20℃ storage conditions.The inhibitory activity of SME was investigated and compared to that of allopurinol.The results obtained with the PAD showed agreement with those obtained with the microplate method.In conclusion,the proposed PAD method is simple,accurate and has a potential for point-of-care testing.It also holds promise for use in rapid quality testing of medicinal herbs,intermediate products,and preparations of traditional Chinese medicines.

A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

Study the effect of kidney stones on serum xanthine oxidase, ecto-5'-nucleotidase activity and E3 SUMO-protein ligase NSE2(NSMCE2) in Malaysian individuals

Objective: To verify possible relations between 5'-nucleotidase, xanthine oxidase to E3 small ubiquitin-like modifier-protein ligase non structural maintenance of chromosomes elements 2 in sera patients with kidney stones and to evaluate the possibility of a new biomarker for the evaluation of kidney damage. Methods: A sixty patients with known kidney stones who appeared the government health clinics in Kuantan–Pahang and fifty apparently healthy were taken as control group. The 5'-nucleotidase,xanthine oxidase and other biochemical parameters were measured by colorimetric tests. The serum NSMCE2 were measured by enzyme linked immunosorbent assay.Results: The mean serum xanthine oxidase [(39.98±19.70) IU/L] and ecto-5'-nucleotidase activity(40.03±9.53 IU/L) were significantly higher than the controls' levels of(18.04 ±6.26) and(16.06 ±4.61) IU/L respectively. There were 85.00% and 83.33%, of patients with kidney stones who had abnormal ecto-5'-nucleotidase activity and uric acid respectively while xanthine oxidase activity was less sensitive 58.33%.Conclusions: The present study suggests that the increase in serum of xanthine oxidase,ecto-5'-nucleotidase activities E3 small ubiquitin-like modifier-protein ligase NSE2 concentration can be used as biomarkers for diagnosis of kidney damage in patients with kidney stone,also in developments of change DNA damage and inflammation disorders in these patients.

Combinatorial co-expression of xanthine dehydrogenase and chaperone XdhC from Acinetobacter baumannii and Rhodobacter capsulatus and their applications in decreasing purine content in food

This study investigated the combinatorial expression of xanthine dehydrogenase(XDH)and chaperone XdhC from Acinetobacter baumannii and Rhodobacter capsulatus and their applications in decreasing purine content in the beer,beef and yeast.Naturally occurring xdhABC gene clusters of A.baumannii CICC 10254 and R.capsulatus CGMCC 1.3366 as well as two refactored clusters constructed by exchanging their xdhC genes were overexpressed in Escherichia coli and purified to near homogeneity.RcXDH chaperoned by AbXdhC showed nearly the same catalytic performance as that by RcXdhC,except for the decreased substrate affinity.While the AbXDH co-expressed with RcXdhC displayed enhanced acidic adaptation but weakened catalytic activity.All the XDHs degraded purines in beer,beef and yeast extract effectively,indicating potential applications in low-purine foods to prevent hyperuricemia and gout.The study also presents a method for exploiting the better chaperone XdhC and novel XDHs by functional complement activity using existing XdhCs such as RcXdhC.

Correlation of docking energies with spectroscopic kinetic assays of potential xanthine oxidase substrates

Here we present a docking model that ranks compounds according to their potential effectiveness as a potential substrate or inhibitor. We utilize xanthine oxidase (XO), a multi-cofactor oxido-reductase which converts hypoxanthine to xanthine and xanthine to uric acid. During the reductive half reaction, electrons flow from the molybdopterin, to each of two Fe/S centers, and finally to FAD. During the oxidative half reaction, electrons are passed from the FAD to O2. Under ideal physiological conditions, this reduction of oxygen generates H2O2 and, under multiple turnover conditions, superoxide in amounts which is regulated by catalase and superoxide dismutase. Utilizing computer modeling predictions of the docking orientations and energies of a group of purine based structures was selected. Correlating computer estimations with steady state kinetic data, a rapid screening process for inhibittor prediction was highlighted. This method allows educated selection of likely inhibitors, thereby decreasing the time and supplies required to complete a traditional kinetic analysis screening. Results demonstrate the functionality and reliability of this method and have proven particularly useful in understanding binding orienttations or poses of each compound.

<i>In Silico</i>and <i>in Vitro</i>Approach for the Understanding of the Xanthine Oxidase Inhibitory Activity of Uruguayan Tannat Grape Pomace and Propolis Poliphenols

The use of food additives with xanthine oxidase (XO) inhibitory activity offers an alternative approach to hyperuricemic and gout disease treatment, and provides an example of antioxidant nutraceutics. The in vitro and in silico XO inhibitory activity of polyphenols from Uruguayan Tannat grape pomaces and propolis extracts was evaluated as well as the scavenging capacity of said compounds. When comparing propolis and grape pomace samples, the in vitro studies demonstrated that polyphenols extracted from propolis are more active as free radical scavengers than those from Tannat grape pomace. Both natural products effectively inhibited XO but the capacity of phenols present in GP is higher than the one present in P. The high content of anthocyanins in GP, absent in P, could account for this observation. In silico assays allowed us to determine relevant ligand-receptor interactions between polyphenols, from a database built with previously reported polyphenols from both natural products, and the active site of XO. The in silico results showed that compound (E)-isoprenylcaffeate from propolis was the best potential XO inhibitor displaying hydrophobic aromatic interaction between the conjugated ring of the caffeate moiety and polar interactions between hydroxyl groups from caffeate with the active site polar residues. Among grape pomaces, the Cyanidin-3-O-(6-(E)-p-coumaroyl)-glucoside was the best XO inhibitor;its moiety oxychromenyl being relevant to the docking stabilization. All these results lead us to propose Uruguayan propolis and Tannat grape pomace extracts as food additives as well as phytopharmaceuticals to decrease the uric acid levels in gout disease and to act against oxidative stress.

Inhibitory Activity on Xanthine Oxidase and Antioxidant Properties of <i>Teucrium polium</i>

The antioxidative activities of subfractions;methanol (CE), chloroform (CHE) and ethyl acetate (EAE) of Teucrium polium extracts (TPE) were investigated. HPLC analysis of the plant revealed the existence of procyanidins B1 and B2, gallic acid, catechin and epicatechin. All the extracts showed inhibitory properties on xanthine oxidase, with IC50 ranging from 0.80 ± 0.07 to 11.76 ± 0.50 μM/quercetin equivalent. In the cellular system, all the extracts showed a protec-tive effect greater than those of quercetin, rutin and gallic acid against t-BHP induced oxidative damages in human erythrocytes. These results were clearly confirmed by a modified thiobarbituric acid-reactive species (TBARS), and β-carotene/linoleic acid assay which demonstrated that CHE possess an inhibition ratio of the linoleic acid oxidation (83.11%) close to that of BHT (96.77%). In addition, the results showed that the extracts possess a potent DPPH radical scavenging activity and gave a reduction power greater than rutin, quercetin, gallic acid and ascorbic acid in FRAP assay. These results show that Teucrium polium extracts have strong antioxidant effects and may have some clinical benefits.

Effect of TongFengNing Decoction on Uric Acid Levels and Xanthine Oxidase Activity in Hyperuricemia Rats

Objective To observe the effect of TongFengNing Decoction （TD） on uric acid levels, xanthine oxidase （XOD） activity, and XOD mRNA expression of hyperuricemia （HUA） model rats. Methods： 90 rats were randomly divided into 6 groups （n=15）, and the HUA model in all groups except the blank group was established by administering hypoxanthine （HX） by gavage and injecting potassium oxonate （OAPS） intraperitoneally. Rats in all TD groups and allopurinol group were administered multiple doses of TD and a single dose of allopurinol by gavage twice daily for 21 days, while the blank group and the model group were administered normal saline. On the 7th, 14th, and 21st days of drug intervention, serum uric acid （SUA）, urine uric acid （UUA）, intestinal uric acid （IUA）, as well as XOD activity and mRNA expression in the liver and small intestine were measured in randomly selected 5 rats of each group. Results： On the 14th and 21st days of intervention, all TD dose groups and the allopurinol group showed decreased SUA and IUA levels, increased UUA levels, as well as decreased XOD activity and mRNA expression in the liver and small intestine, compared with the model group （P 〈 0.05）. The low- and high-dose TD group and the allopurinol group showed increased SUA and IUA levels, as well as XOD activity and mRNA expression in the liver and small intestine, and decreased UUA levels, compared with the moderate-dose TD group （P〈0.05）. Upon extending the drug intervention time of each TD dose group, SUA and IUA levels, XOD activity, and XOD mRNA expression in the liver and small intestine decreased and UUA levels increased （P 〈 0.05）. Conclusion： TD reduces SUA levels in HUA model rats, which promotes uric acid excretion and inhibits XOD activity and XOD mRNA expression to reduce uric acid production. The reduction in uric acid level by the intermediate dose of TD was better than that by allopurinol and the low and high doses of TD.

Inhibition of xanthine oxidase alleviated pancreatic necrosis via HIF-1α-regulated LDHA and NLRP3 signaling pathway in acute pancreatitis

Acute pancreatitis(AP)is a potentially fatal condition with no targeted treatment options.Although inhibiting xanthine oxidase(XO)in the treatment of AP has been studied in several experimental models and clinical trials,whether XO is a target of AP and what its the main mechanism of action is remains unclear.Here,we aimed to re-evaluate whether XO is a target aggravating AP other than merely generating reactive oxygen species that trigger AP.We first revealed that XO expression and enzyme activity were significantly elevated in the serum and pancreas of necrotizing AP models.We also found that allopurinol and febuxostat,as purine-like and non-purine XO inhibitors,respectively,exhibited protective effects against pancreatic acinar cell death in vitro and pancreatic damage in vivo at different doses and treatment time points.Moreover,we observed that conditional Xdh overexpression aggravated pancreatic necrosis and severity.Further mechanism analysis showed that XO inhibition restored the hypoxia-inducible factor 1-alpha(HIF-1α)-regulated lactate dehydrogenase A(LDHA)and NOD-like receptor family pyrin domain containing 3(NLRP3)signaling pathways and reduced the enrichment of^(13)C_(6)-glucose to^(13)C_(3)-lactate.Lastly,we observed that clinical circulatory XO activity was significantly elevated in severe cases and correlated with C-reactive protein levels,while pancreatic XO and urate were also increased in severe AP patients.These results together indicated that proper inhibition of XO might be a promising therapeutic strategy for alleviating pancreatic necrosis and preventing progression of severe AP by downregulating HIF-1α-mediated LDHA and NLRP3 signaling pathways.

Electrocatalytic Oxidation and Simultaneous Determination of Uric Acid,Xanthine,Hypoxanthine and Dopamine Based on β-Cyclodextrin Modified Glassy Carbon Electrode

A novel covalently modified glassy carbon electrode with β-cyclodextrin was prepared via electropolymerization technique for the simultaneous determination of uric acid（UA）,xanthine（XA）,hypoxanthine（HX） and dopamine（DA）.This new electrode presented an excellent electrocatalytic activity towards the oxidation of UA,XA,HX and DA by cyclic voltammetry（CV） method.The oxidation peaks of the four compounds were well defined and had the enhanced peak currents.The separation potentials of the oxidation peaks for DA-UA,UA-XA and XA-HX were 150,390 and 360 mV in CV,respectively.By means of differential pulse voltammetry（DPV） method,the calibration curves in the ranges of 10―225,5―105,10―170 and 5―150 μmol/L were obtained for UA,XA,HX and DA,respectively.The lowest detection limits（S/N=3） were 5,1.25,5 and 1.5 μmol/L for UA,XA,HX and DA,respectively.The practical application of the modified electrode was demonstrated by the determination of DA in hydrochloride injection and UA,XA,HX in human urine samples.

A rice XANTHINE DEHYDROGENASE gene regulates leaf senescence and response to abiotic stresses

Xanthine dehydrogenase, a member of the molybdenum enzyme family, participates in purine metabolism and catalyzes the generation of ureides from xanthine and hypoxanthine. However, the mechanisms by which xanthine dehydrogenase affects rice growth and development are poorly understood. In the present study, we identified a mutant with early leaf senescence and reduced tillering that we named early senescence and less-tillering 1(esl1). Map-based cloning revealed that ESL1 encodes a xanthine dehydrogenase, and it was expressed in all tissues. Chlorophyll content was reduced and chloroplast maldevelopment was severe in the esl1 mutant. Mutation of ESL1 led to decreases in allantoin, allantoate, and ABA contents. Further analysis revealed that the accumulation of reactive oxygen species in esl1 resulted in decreased photosynthesis and impaired chloroplast development, along with increased sensitivity to abscisic acid and abiotic stresses. Ttranscriptome analysis showed that the ESL1 mutation altered the expression of genes involved in the photosynthesis process and reactive oxygen species metabolism.Our results suggest that ESL1 is involved in purine metabolism and the induction of leaf senescence.These findings reveal novel molecular mechanisms of ESL1 gene-mediated plant growth and leaf senescence.

Electrochimical determination of uric acid, xanthine and hypoxanthine by poly(xylitol) modified glassy carbon electrode

The novel covalently modified glassy carbon electrode with poly(xylitol) was prepared using an electropolymerization technique for the simultaneous determination of uric acid(UA), xanthine(XA) and hypoxanthine(HX). This new electrode presents an excellent electrocatalytic activity towards the oxidation of UA, XA and HX by cyclic voltammetry(CV) method. The oxidation peaks of the three compounds were well defined and had enhanced the peak currents. The separation potentials of the oxidation peak potentials for UA-XA and XA-HX were 380 and 370 mV in CV, respectively. Using differential pulse voltammetry(DPV) method, the calibration curves in the ranges of 5-55, 1.3-75.3 and 4-59 μmol/L were obtained for HX, XA and UA, respectively. The lowest detection limits(S/N=3) were 4.5, 0.75 and 3.75 μmol/L for HX, XA and UA, respectively. The practical application of the modified electrode was demonstrated by the determination of UA, XA, HX in human urine samples.

Purification and Characterisation of Xanthine Oxidoreductases from Local Bovids in Malta

Xanthine oxidoreductase (XOR) is a molybdoflavoprotein mainly involved in purine catabolism. It exists in two forms, the oxidase (XO) and dehydrogenase (XDH) which are inter-convertible within mammalian cells. Although various researchers have reported the extraction of mammalian XOR, no extractions have yet been carried out in Malta and subsequently no characterizations are available. In this study, XOR was successfully purified from bovine, caprine and ovine milk through a multistep purification process involving both chemical and chromatographic techniques. The molecular weights of the native enzyme were found to be 295 kDa, 281 kDa and 275 kDa, representing the bovine, caprine and ovine XOR respectively. Western blot showed XOR to be represented on SDS-PAGE by a minimum of three major bands having molecular weights of 151 kDa, 131 kDa and 85 kDa. While all samples showed activity on native PAGE, spectrophotometric assays revealed the bovine XOR to be the most active. Surprisingly, the addition of NAD+ to the assay mixture inhibited enzyme activity of the bovine and caprine XOR whereas the ovine XOR doubled its activity in response to NAD+. The latter also showed a lower binding affinity to heparin. Following incubation with trypsin, XOR was irreversibly converted to its oxidase form in all samples as reflected by the observed increase in XO activity.

Biological evaluation of 8-alkyl xanthines as potential cytotoxic agents

A series of 8-substituted alkyl xanthines were evaluated in vitro to test the cytotoxocity in cells. For this experiment, we utilized different mammalian cancer cell lines primarily representing prostrate and lung. One of the compounds synthesized, viz. 8-tertbutyl caffeine showed potent anticancer activity at low concentrations against DU145 when compared to adriamycin. Further experiments were carried out to check the cell cycle arrest in the DU145 cells treated with adriamycin, caffeine and 8-tert butyl caffeine. We observed that there was an arrest in G1 phase of cell cycle at 24 hours while at 48 hours of incubation, the cells were constantly distributed (59.71% -70.79%). We conclude that the effect of 8-tertbutyl caffeine is relatively comparable to caffeine whereas in adriamycin treated cells, we observed the cells underwent G2 arrest. We evaluate the studies on these effects by showing potent analogues which could be used as promising anticancer agents.

Poly Ethylene Glycols (PEG) and Micelles as Efficient Catalysts for the Oxidation of Xanthine Derivatives under Conventional and Non-Conventional Conditions

Oxidation of Xanthine alkaloid have been studied with various one and two electron oxidizing agents using PEGs and micelle forming surfactants. The reaction is too sluggish in solution phase, but moderately fast in presence of PEGs and micelles. However, the reactions are dramatically enhanced under microwave irradiations. Present protocol has several advantages, such as solvent-free conditions, during work-up, fast reaction times, high yields, eco-friendly operational and experimental simplicity, readily available additives as catalysts.

Determination of Hypoxanthine in the Presence of Copper by Adsorptive Stripping Voltammetry

A stripping method for the determination of hypoxanthine in the presence of copper at the submicromolar concentration levels is described. The method is based on controlled adsorptive accumulation of hypoxanthine-copper at the thin-film mercury electrode followed by a fast linear scan voltammetric measurement of the surface species. Optimum experimental conditions were found to be the use of 1.0 × 10﹣3 mol·L﹣1 NaOH solution as electrolyte supporting, an accumulation potential of ﹣0.50 V and a linear scan rate of 200 mV·s﹣1. The response of hypoxanthine-copper is linear over the concentration ranges of 10 - 60 ppb. For an accumulation time of 30 minutes, the detection limit was found to be 250 ppt (1.8 × 10﹣9 mol·L﹣1). Adequate conditions for measuring the hypoxanthine in the presence of metal ions, xanthine, uric acid and other nitrogenated bases were also investigated. The utility of the method is demonstrated by the presence of hypoxanthine associated in ATP or ssDNA.