阅读说明：本技术 判断肠道内肠碱性磷酸酶活性水平的检测方法及相关应用 (Detection method for judging activity level of intestinal alkaline phosphatase in intestinal tract and related application ) 是由 高辰哲 李傲辰 徐庆 李鑫荣 韩建春 惠觅宙 于 2020-02-13 设计创作，主要内容包括：本发明公开了一种判断肠道内肠碱性磷酸酶活性水平的检测方法及相关应用,属于检测领域。本发明通过尿液pH检测来间接判断胃肠道酸碱负荷水平和肠道内pH相关肠碱性磷酸酶活性水平,胃肠道酸碱负荷和尿液pH呈正相关的关系,肠道内碱负荷时与肠碱性磷酸酶活性水平呈正相关,并可判断通过服用肠碱化制剂在治疗肠道碱性磷酸酶活性相关疾病的食用剂量和疗效。本研究还表明通过服用肠碱化制剂(例如：含有微胶囊包膜丁酸钠的肠碱化和脂肪吸收阻断矿物质爆膨颗粒)治疗后尿液pH达到6.4或以上时的剂量有利于治疗肠道碱性磷酸酶活性相关疾病(高血糖、高血脂和结肠炎)的治疗。本发明通过尿液pH检测快捷、简单有效。(The invention discloses a detection method for judging the activity level of intestinal alkaline phosphatase in an intestinal tract and related application, and belongs to the field of detection. According to the invention, the acid-base load level of the gastrointestinal tract and the pH-related intestinal alkaline phosphatase activity level in the intestinal tract are indirectly judged through urine pH detection, the acid-base load of the gastrointestinal tract and the urine pH are in a positive correlation, the alkali load in the intestinal tract and the intestinal alkaline phosphatase activity level are in a positive correlation, and the edible dosage and the curative effect of treating the intestinal alkaline phosphatase activity-related diseases by taking the intestinal alkalizing preparation can be judged. The study also shows that the dose at which the urine pH reaches 6.4 or above after treatment by taking an enteroalkalizing agent (e.g., enteroalkalizing and fat absorption blocking mineral bursting pellets containing microcapsule-coated sodium butyrate) is beneficial for treating diseases related to the intestinal alkaline phosphatase activity (hyperglycemia, hyperlipidemia and colitis). The invention is fast, simple and effective in urine pH detection.)

1. A detection method for judging the activity level of intestinal alkaline phosphatase in an intestinal tract is characterized in that the acid-base load level of the gastrointestinal tract and the activity level of intestinal alkaline phosphatase related to the pH in the intestinal tract are indirectly judged through urine pH detection, the acid-base load of the gastrointestinal tract and the pH of urine are in a positive correlation, and the alkali load in the intestinal tract and the activity level of intestinal alkaline phosphatase are in a positive correlation.

2. The assay of claim 1, wherein the intestinal alkaline phosphatase activity is an activity of inactivating endotoxin, inhibiting secretion of inflammatory factor TNF-alpha by human blood leukocytes and vascular endothelial cells, and inhibiting removal of human leukocytes.

3. The assay for determining the level of intestinal alkaline phosphatase activity according to claim 1 or 2, wherein the intestinal alkaline phosphatase activity is enhanced by an intestinal alkalizing agent capable of altering the urine pH and induced by the intestinal alkaline phosphatase expression promoter sodium butyrate.

4. The assay for determining the activity level of intestinal alkaline phosphatase in the intestinal tract according to claim 3, wherein the intestinal alkalizing agent is an intestinal alkalizing and fat absorption blocking mineral burst granular preparation containing microcapsule coated sodium butyrate;

or the intestinal alkalization preparation is a mineral burst swelling granular preparation and a microcapsule coated sodium butyrate coated by a capsule or an enteric coated tablet, wherein the intestinal alkalization preparation is intestinal alkalization and fat absorption blocking;

or the intestinal alkalizing preparation is an intestinal alkalizing and fat absorption blocking mineral medicine or intestinal alkalizing mineral food containing microcapsule coated sodium butyrate;

or the intestinal alkalizing preparation is a mineral drug for blocking intestinal alkalization and fat absorption, or intestinal alkalizing mineral food and capsule, or enteric capsule or enteric coated tablet wrapped microcapsule coated sodium butyrate.

5. The test method for judging the dosage and the curative effect of the compound intestinal alkalization preparation for treating the diseases related to the reduction of the activity of the intestinal alkaline phosphatase is characterized in that the dosage and the curative effect of the intestinal alkalization preparation for treating the diseases related to the reduction of the activity of the intestinal alkaline phosphatase are judged by urine pH detection.

6. The test method according to claim 5, wherein the pH of morning urine of the patient with the disease associated with decreased intestinal alkaline phosphatase activity taking the preparation is measured by: when the urine pH is less than or equal to 5.5, an intestinal alkalizing preparation is required to treat diseases related to the reduction of the activity of the intestinal alkaline phosphatase; if the pH value of urine is not less than 6.5, the intestinal alkalization preparation is judged to play a role in treating diseases related to the reduction of the activity of the intestinal alkaline phosphatase.

7. The assay of claim 5 or 6, wherein the disease associated with decreased intestinal alkaline phosphatase activity comprises hyperglycemia, hyperlipidemia and/or colitis.

8. The inspection method according to any one of claims 5 to 7,

the intestinal alkalization preparation is an intestinal alkalization and fat absorption blocking mineral burst expansion granular preparation containing microcapsule coated sodium butyrate;

or the intestinal alkalization preparation is a mineral burst swelling granular preparation and a microcapsule coated sodium butyrate coated by a capsule or an enteric coated tablet, wherein the intestinal alkalization preparation is intestinal alkalization and fat absorption blocking;

or the intestinal alkalizing preparation is an intestinal alkalizing and fat absorption blocking mineral medicine or intestinal alkalizing mineral food containing microcapsule coated sodium butyrate;

or the intestinal alkalizing preparation is a mineral drug for blocking intestinal alkalization and fat absorption, or intestinal alkalizing mineral food and capsule, or enteric capsule or enteric coated tablet wrapped microcapsule coated sodium butyrate.

9. The test method for judging the dosage and curative effect of the intestinal alkalization preparation for treating the diseases related to the human leukocyte TNF-alpha secretion increase of the intestinal mucosa is characterized in that the dosage and curative effect of the intestinal alkalization preparation for treating the diseases related to the human leukocyte TNF-alpha secretion increase of the intestinal mucosa are judged by urine pH detection.

10. The method according to claim 9, wherein the pH of morning urine of the patient with the disease associated with increased human leukocyte TNF- α secretion of the intestinal mucosa to which the preparation is administered is measured, and the pH of urine is greater than or equal to 6.5, so that the preparation is determined to be effective in treating the disease associated with increased human leukocyte TNF- α secretion of the intestinal mucosa.

11. The test method according to claim 9 or 10, wherein the disease associated with increased secretion of human leukocyte TNF- α from the intestinal mucosa comprises colitis.

12. The inspection method according to any one of claims 9 to 11,

the intestinal alkalization preparation is an intestinal alkalization and fat absorption blocking mineral burst expansion granular preparation containing microcapsule coated sodium butyrate;

or the intestinal alkalization preparation is a mineral burst swelling granular preparation and a microcapsule coated sodium butyrate coated by a capsule or an enteric coated tablet, wherein the intestinal alkalization preparation is intestinal alkalization and fat absorption blocking;

or the intestinal alkalizing preparation is an intestinal alkalizing and fat absorption blocking mineral medicine or intestinal alkalizing mineral food containing microcapsule coated sodium butyrate;

or the intestinal alkalizing preparation is a mineral drug for blocking intestinal alkalization and fat absorption, or intestinal alkalizing mineral food and capsule, or enteric capsule or enteric coated tablet wrapped microcapsule coated sodium butyrate.

Technical Field

The invention relates to the field of detection, in particular to a detection method for judging the activity level of intestinal alkaline phosphatase in an intestinal tract and related application.

Background

Type 2 diabetes is a chronic metabolic disease that occurs when tissues fail to produce sufficient insulin and do not use insulin effectively (insulin resistance). Tissue insulin resistance and insufficient production of insulin lead to prolonged elevation of blood glucose levels, which adversely affect the eyes, heart, kidneys and feet and are referred to as diabetic complications (1). Type 2 diabetes is often accompanied by hyperlipidemia, hypertension, and obesity, known as metabolic syndrome. Thus, type 2 diabetes is typically part of the metabolic syndrome (2).

Recent clinical studies of a large sample of people show that the urine pH of patients with metabolic syndrome (hypertension, hyperlipidemia and hyperglycemia) is generally less than 5.5, and the method suggests that acid urine is a reliable method for diagnosing metabolic syndrome and intestinal leakage by using a urine sample (3, 4); there is also literature indicating that decreased alkaline phosphatase activity in the gut is a cause of hyperglycemia, hyperlipidemia and colitis (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). The activity of intestinal alkaline phosphatase is mainly dependent on the pH level, under alkaline pH conditions intestinal alkaline phosphatase is active. The traditional method for detecting alkaline phosphatase is to sample and detect the alkaline phosphatase through excrement, and the detection reagent and the detection method have relatively high cost and are not beneficial to popularization.

Reference to the literature

1.Type 2Diabetes Mellitus:A Review of Current TrendsType 2DiabetesMellitus:A Review of Current Trends.Abdulfatai B.Olokoba,1,*OlusegunA.Obateru,2and Lateefat B. Olokoba3

2.A Comprehensive Review on Metabolic Syndrome.Jaspinder Kaur*Authorinformation Article notes Copyright and License information Disclaimer

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4.Cho Y H,Lee S Y,Jeong D W,et al.The Association between a Low UrinepH and the Components of Metabolic Syndrome in the Korean Population:FindingsBased on the 2010 Korea National Health and Nutrition Examination Survey[J].Journal of Research in Medical Sciences,2014,19(7):599-604.

5.Kaooru Geddes and Dana J Philpott(2008),Anew role for intestinalalkaline phosphatase in gut barrier maintenance,Gastroenterology,135:8-12.

6.Jean-Paul Lalles(2014),Luminal ATP:the missing link betweenintestinal alkaline phosphatase,the gut microbiota,and inflammation？Am JPhysiol Gastrointest Liver Physiol, 306:G824-G825.

7.Jean-Paul Lalles(2016),Intestinal alkaline phosphatase:novelfunctions andprotective effects,Nutrition Reviews,72(2):82-94.

8.Mehrbodet al(2014),interplay between intestinal alkalinephosphatase,diet,gut microbes and immunity,World Journal of Gastroenterology,20(42):15650-15656.

9.Cani et al(2007),metabolic endotoxemia initiates obesity andinsulin resistance, Diabetes,56:1761-1772.

10.Jason Fawley and David M.Gourlay(2016),Intestinal alkalinephosphatase:a summary of its role in clinical disease,Journal of SurgicalResearch,202(1):1225-1234.

11.Kiffe-Moreira and Milan et al(2014),Catalytic signatureof a heat-stable,chimeric human alkaline phosphatase with therapeutic potential,PlosOne，9(2):e89374.

12.Takanari Nakano,Ikuo Inoue,Iwao Koyama,Kenta Kanazawa,Koh-ichiNakamura,Sonoko Narisawa and Al(2007),Disruption of the murine intestinalalkaline phosphatase gene Akp3 impairs lipid transcytosis and inducesvisceral fat accumulation and hepatic steatosis.Am J Physiol GastrointestLiver Physiol 292:G1439-G1449.

13.Jean-Paul Lalles(2015),Intestinal alkaline phosphatase in stool:Anovel biomarker for metabolic diseases,EBioMedicine,2:1866.

14.Sonoko Narisawa,Lei Huang,Arata Iwasaki,Hideaki Hasegawa,David H.Alpers,and Jose Luis Millan(2003),Accelerated Fat Absorption in IntestinalAlkaline Phosphatase Knockout Mice.MOLECULAR AND CELLULAR

BIOLOGY,23(21):7525–7530.

15.Kanakaraju Kaliannan,Sulaiman R.Hamarneh,KonstantinosP.Economopoulos,Sayeda Nasrin Alam,Omeed Moaven,Palak Patel,Nondita S.Malo,Madhury Ray,M.Abtahi,Nur Muhammad,Atri Raychowdhury,Abeba Teshager,MussaM.Rafat Mohamed,Angela K.Rizwan Ahmed,Shahrad Hakimian,Sonoko Narisawa,JoseLuis Millan, Elizabeth Hohmann,H.Shaw Warren,Atul K.Bhan,Madhu S.Malo,andRichard A.Hodin(2013),Intestinal alkaline phosphatase prevents metabolicsyndrome in mice, PNAS,110(17):7003-7008.

16.Malo M S.AHigh Level of Intestinal Alkaline Phosphatase IsProtective against Type 2Diabetes Mellitus Irrespective of Obesity[J].Ebiomedicine,2015,2(12):2016-2023.

17.Poelstra K,BakkerWW,Klok PA,Hardonk MJ,Meijer DK(1997),Aphysiologic function for alkaline phosphatase:endotoxin detoxification.LabInvest,76:319–327.

18.Poelstra K,Bakker WW,Klok PA,Kamps JA,Hardonk MJ,Meijer DK(1997),Dephosphorylation of endotoxin by alkaline phosphatase in vivo,Am JPathol.151(4):1163-9.

19.Bentala H,Verweij WR,Huizinga-Van der Vlag A,van Loenen-WeemaesAM, Meijer DK,Poelstra K(2002),Removal of phosphate from lipid A as astrategy to detoxify lipopolysaccharide,Shock,18(6):561-6.

20.Jennifer et al(2007),Intestinal alkaline phosphatase detoxifieslipopolysaccharide and prevents inflammation in response to the gutmicrobiota,Cell Host Microbe, 2(6):371-382.

21.Beumer et al(2003)Calf intestinal alkaline phosphatase,a noveltherapeutic drug for lipopolysaccharide(LPS)-mediated diseases,attenuates LPStoxicity in mice and piglets, 307(2):737-744.

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Disclosure of Invention

The invention aims to provide a detection method capable of rapidly judging the activity level of intestinal alkaline phosphatase in an intestinal tract and related application, and an effect test method for judging the effect of an intestinal alkalization preparation (containing microcapsule coated sodium butyrate (or microcapsule coated sodium butyrate jointly used by capsules, enteric capsules or enteric coated tablets) on treating the metabolic disease related to alkaline phosphatase in the process of intestinal alkalization and fat absorption blocking mineral burst-swelling granules) by detecting the pH change of urine.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

on one hand, the invention provides a detection method for judging the activity level of intestinal alkaline phosphatase in an intestinal tract, which indirectly judges the acid-base load level of the gastrointestinal tract and the activity level of intestinal alkaline phosphatase related to the pH in the intestinal tract by urine pH detection, wherein the acid-base load of the gastrointestinal tract and the pH of urine are in a positive correlation, and the acid-base load in the intestinal tract and the activity level of intestinal alkaline phosphatase are in a positive correlation.

Determining gastrointestinal acid-base load by urine pH detection, and determining alkaline phosphatase activity level: the level of alkaline phosphatase activity in the intestine is greater when the urine pH is alkaline than when the pH is acidic.

The inventors of the present invention previously hypothesized that the acidic urinary pH (4.5-5.5) in patients with metabolic syndrome (hypertension, hyperlipidemia, hyperglycemia) may be due to an excessive acid load in the gastrointestinal tract, i.e. eating too much of a high-fat and high-protein food that promotes gastric acid secretion. In order to research the correlation between acid and alkaline foods and intestinal acid-base load and urine pH, a method for judging the intestinal acid-base load and the intestinal alkaline phosphatase activity level by using urine pH measurement is found, the intestinal acid-base load is judged by the urine pH, when the urine pH is alkaline, the intestinal alkaline load is higher, so that the alkaline phosphatase activity in the intestinal tract is indirectly shown to be at a higher level, and the method has an important guiding effect on treating diseases related to the intestinal alkaline phosphatase activity, such as hyperglycemia, hyperlipidemia, colitis and the like, for example, the alkaline bulking in the intestinal tract is improved by taking an intestinal alkalizing preparation (containing micro-capsule coated sodium butyrate (or micro-capsule coated sodium butyrate jointly coated with capsules or enteric coated tablets) and blocking the mineral burst particles for fat absorption), so that the alkaline phosphatase is at a higher activity level, thereby playing a role in treating diseases related to the activity of the alkaline phosphatase in the intestinal tract.

Further, the intestinal alkaline phosphatase activity is an activity of inactivating endotoxin, inhibiting secretion of inflammatory factor TNF-alpha by human blood leukocytes and vascular endothelial cells, and inhibiting removal of human leukocytes. The activity of alkaline phosphatase is judged according to the pH of urine, so that the activities of inactivating endotoxin, inhibiting the secretion of inflammatory factor TNF-alpha by human blood leukocyte and vascular endothelial cell and inhibiting the removal of human leukocyte are judged.

Further, the intestinal alkaline phosphatase activity is induced and enhanced by an intestinal alkalizing agent capable of altering the urine pH.

Further, the intestinal alkalization preparation is an intestinal alkalization and fat absorption blocking mineral burst granular preparation containing microcapsule coated sodium butyrate.

Further, the intestinal alkalization preparation is an intestinal alkalization and fat absorption blocking mineral burst swelling granular preparation and microcapsule coated sodium butyrate wrapped by capsules or enteric coated tablets.

Further, the intestinal alkalizing preparation is an intestinal alkalizing and fat absorption blocking mineral drug or an intestinal alkalizing mineral food containing microcapsule coated sodium butyrate.

Further, the intestinal alkalizing preparation is an intestinal alkalizing and fat absorption blocking mineral medicament or an intestinal tract alkalizing mineral food and a microcapsule coated sodium butyrate wrapped by a capsule or an enteric coated tablet. .

The urine pH is used for judging the intestinal alkaline phosphatase activity level, so that the alkaline phosphatase activity is improved by increasing the intestinal alkaline load by taking the intestinal alkalization preparation, the urine pH is increased, and the edible dosage and the curative effect of treating the related diseases with the decreased alkaline phosphatase activity by taking the intestinal alkalization preparation are judged.

In another aspect, an examination method for determining the dosage and efficacy of an intestinal alkalizing preparation for treating diseases related to decreased intestinal alkaline phosphatase activity is provided, wherein the dosage and efficacy of the intestinal alkalizing preparation for treating diseases related to decreased intestinal alkaline phosphatase activity is determined by urine pH detection.

Further, by measuring the morning urine pH of a patient with a disease associated with decreased intestinal alkaline phosphatase activity taking the preparation: when the urine pH is less than or equal to 5.5, an intestinal alkalizing preparation is required to treat diseases related to the reduction of the activity of the intestinal alkaline phosphatase; if the pH value of urine is not less than 6.5, the intestinal alkalization preparation is judged to play a role in treating diseases related to the reduction of the activity of the intestinal alkaline phosphatase.

Further, the diseases associated with decreased intestinal alkaline phosphatase activity include hypertension, hyperlipidemia and/or hyperglycemia, which also belong to metabolic syndrome.

Further, the intestinal alkalization preparation is an intestinal alkalization and fat absorption blocking mineral burst granular preparation containing microcapsule coated sodium butyrate.

Further, the intestinal alkalization preparation is an intestinal alkalization and fat absorption blocking mineral burst swelling granular preparation and microcapsule coated sodium butyrate wrapped by capsules or enteric coated tablets.

Further, the intestinal alkalizing preparation is an intestinal alkalizing and fat absorption blocking mineral drug or an intestinal alkalizing mineral food containing microcapsule coated sodium butyrate.

Further, the intestinal alkalizing preparation is an intestinal alkalizing and fat absorption blocking mineral medicament or an intestinal tract alkalizing mineral food and a microcapsule coated sodium butyrate wrapped by a capsule or an enteric coated tablet.

The dose can be 33 g.

In another aspect, the test method for judging the dosage and curative effect of the intestinal alkalization preparation for treating the diseases related to the human leukocyte TNF-alpha secretion increase of the intestinal mucosa is provided, and the dosage and curative effect of the intestinal alkalization preparation for treating the diseases related to the human leukocyte TNF-alpha secretion increase of the intestinal mucosa are judged by urine pH detection.

Further, by detecting the pH value of morning urine of a patient with a disease related to the increase of human leukocyte TNF-alpha secretion of the intestinal mucosa after taking the intestinal alkalization preparation, if the pH value of the urine is not less than 6.5, the intestinal alkalization preparation is judged to play a role in treating the disease related to the increase of human leukocyte TNF-alpha secretion of the intestinal mucosa.

Further, the diseases related to the increase of human leukocyte TNF-alpha secretion of intestinal mucosa include, but are not limited to, colitis.

Further, the intestinal alkalization preparation is an intestinal alkalization and fat absorption blocking mineral burst granular preparation containing microcapsule coated sodium butyrate.

Further, the intestinal alkalization preparation is an intestinal alkalization and fat absorption blocking mineral burst swelling granular preparation and microcapsule coated sodium butyrate wrapped by capsules or enteric coated tablets.

Further, the intestinal alkalizing preparation is an intestinal alkalizing and fat absorption blocking mineral drug or an intestinal alkalizing mineral food containing microcapsule coated sodium butyrate.

Further, the intestinal alkalizing preparation is an intestinal alkalizing and fat absorption blocking mineral medicament or an intestinal tract alkalizing mineral food and a microcapsule coated sodium butyrate wrapped by a capsule or an enteric coated tablet. .

The intestinal alkalizing agent can be administered in a dose of 33 g.

The fat absorption blocking mineral may be montmorillonite powder, or hydrotalcite, oat flour, flavoring agent, etc.

Because the taste of the microcapsule coated sodium butyrate is poor, the taste can be improved by coating the microcapsule coated sodium butyrate by capsules or enteric coating sheets.

In yet another aspect, the present invention provides a method for treating a disease associated with decreased alkaline phosphatase activity in the intestinal tract by administering enteric alkalization and fat absorption blocking mineral burst particles comprising microencapsulated sodium butyrate (or microencapsulated sodium butyrate encapsulated in a capsule or enteric coated tablet in combination) to treat diabetes, increase urine pH, and treat a disease associated with decreased alkaline phosphatase activity in the intestinal tract.

After adopting such design, the invention has at least the following advantages:

the invention judges the pH value of the intestinal tract and the activity level of the intestinal alkaline phosphatase by urine pH detection, is quick, simple and effective, and particularly emphasizes that the pH value of the intestinal tract can be judged by the urine pH to judge the activity level of the intestinal alkaline phosphatase, the effect of the intestinal alkaline medicament and food for enhancing the activity of the intestinal alkaline phosphatase is further judged, and the curative effect detection and the application of the intestinal alkalizing preparation (for example, intestinal alkalizing and fat absorption blocking mineral substance explosion-swelling granules containing microcapsule coated sodium butyrate (or microcapsule coated sodium butyrate jointly coated by capsules, enteric capsules or enteric coated tablets) on treating diseases related to the activity of the intestinal alkaline phosphatase, such as hyperglycemia, hyperlipidemia and colitis are realized.

Drawings

The foregoing is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and the detailed description.

FIG. 1 is a schematic diagram of a comparison example of the pH value of urine after acidic food and alkaline food are eaten by healthy participants according to the study of the invention;

FIG. 2 is a schematic diagram of a comparison example of the pH value of urine of a participant of the non-healthy metabolic syndrome after eating acidic food and alkaline food;

FIG. 3 is a schematic diagram of an embodiment of the present invention for investigating the effect of different pH on the LPS inactivation effect of recombinant intestinal alkaline phosphatase;

FIG. 4 is a schematic diagram of an example of the present invention for studying the effect of different endotoxin doses on inducing TNF- α release from human peripheral blood leukocytes and HUVECs;

FIG. 5 is a graphical representation of the removal of human neutrophils from the in-to-out of the gel droplet culture medium studied for the inhibition of intestinal alkaline phosphatase in accordance with the present invention;

FIG. 6 is a graph showing the removal of human mononuclear cells from the in-to-out of the medium in which alkaline phosphatase inhibition was studied according to the present invention.

Detailed Description

Exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention can be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.