JP2014102186A - Nano material diffusion evaluation device, evaluation method, and negative pressure generator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device and a method for quickly evaluating a diffusion condition of a nano material in a lung, particularly whether it reaches an alveolar region depth, and whether the nano material which has reached the alveolar region depth breaks through the lung and a thoracic cavity and leaks out to the thoracic cavity.SOLUTION: A nano material diffusion evaluation device includes a lung installation container for installing both lungs from a trachea and bronchus taken out from an animal, and a negative pressure generator for making inside of the lung installation container in negative pressure. The negative pressure generator can repeatedly make the inside of the lung installation container in negative pressure and atmospheric pressure alternately.

Description

  The present invention relates to a nanomaterial diffusion evaluation apparatus, an evaluation method, and a negative pressure generation apparatus, and more particularly, an apparatus for ex vivo evaluation of diffusion of natural and synthetic nanomaterials to the lungs and pleura, an evaluation method, and the aforementioned The present invention relates to a negative pressure generator used in an apparatus for evaluation.

In recent years, with the advancement of nanotechnology, which is a technology for freely controlling materials in the nanometer (1 nm = 10 ^-9 m) region, that is, the atomic or molecular scale, new materials have been developed on the nanometer scale, Development of devices of such scale is underway.

  A nanomaterial is literally a very small material on the nanoscale, and if it is a material of the same material, weight, and volume, the surface area becomes much wider, so the reactivity becomes very large. Therefore, if nanomaterials are used for conventional products and their parts, it is beneficial in various respects such as miniaturization and high performance. For example, pharmaceuticals, cosmetics, food, food container packaging, textiles, household goods, miscellaneous goods, sports goods Applications to home appliances, electrical and electronic products, paints and inks are expanding. In particular, a carbon nanotube (hereinafter sometimes referred to as “CNT”), which is a material representing nanotechnology, is a synthetic fibrous new material composed of only carbon atoms, and was discovered in 1991. CNT has many excellent characteristics such as light weight, high strength, high thermal conductivity, and a conductor / semiconductor.

  On the other hand, the number of workers engaged in manufacturing or handling is expected to increase rapidly as nanomaterials expand. As for nanomaterials, the composition unit is so small that even a material that has already been confirmed to be safe may exhibit properties different from conventional ones, and there is a growing concern about the impact on the living body.

  For example, in 2008, it was reported that malignant mesothelioma occurs when multi-wall CNT is administered intraperitoneally to p53 hetero knockout mice (see Non-Patent Document 1). As a result, it was suggested that multi-walled CNTs have the same pharmacokinetics as asbestos, which has already been proven to be carcinogenic, and there has been doubt about the safety of CNTs.

  For this reason, the inventors reported that the diameter and rigidity of multi-layer CNTs are extremely related to carcinogenesis in 2011 (see Non-Patent Document 2), and are calling attention to the characteristics to be noted of CNTs. . On the other hand, the Nanotechnology Special Committee (TC229) established in 2005 by the ISO (International Organization for Standardization) standardizes the effects of nanomaterials on health and the environment. The OECD is also making ongoing efforts.

  However, as described above, it has been reported that malignant mesothelioma occurs when intraperitoneal administration of multi-walled CNT (see Non-Patent Document 1), but multi-walled CNT is generally estimated as the test method. In order to evaluate the safety, it is necessary to conduct more detailed research on the long-term persistence in vivo in animals. There is a problem with it.

  In addition, there is a paper in which multi-walled CNTs are administered to the trachea of rats and the effect on the lungs is reported (see Non-Patent Document 3). However, in the experiment described in Non-Patent Document 3, multi-walled CNT is administered to a living rat, and after administration, 3 to 15 days for confirming the onset of inflammation and 60 days for confirming the onset of fibrosis. After raising the rat, it is necessary to take out the tissue and evaluate it, and there is a problem that it takes a very long time to confirm the safety of the multilayer CNT.

  Furthermore, a paper is also known in which multi-walled CNTs are aspirated from the nose of rats and their effects on lungs and toxicity are examined (see Non-Patent Document 4). However, in the experiment described in Non-Patent Document 4, it is necessary to raise the rat for about 6 months after sucking the multilayer CNT from the nose, and then take out the tissue for evaluation. Similar to the above, there is a problem that it takes a very long time to confirm the safety of the multilayer CNT. In addition, rats were exposed for 90 days (6 hours / day, 5 days / one week, 13 weeks in total) in a chamber filled with multi-layer CNT aerosol, and the multi-wall CNTs were aspirated from the nose, causing toxicity to the lungs. The research paper is also known (see Non-Patent Document 5), but there is a problem that it takes a very long time to confirm the safety as in the above-mentioned document.

  By the way, although asbestos is a natural nanomaterial, the occurrence of malignant mesothelioma has been a problem in exposed people since the 1960s. So far, toxicologists have been the main actors in analyzing unexplained risks of chemical substances, but the analysis is Ames test, chromosome aberration test, micronucleus test and animal test usually up to 13 weeks . However, the incubation period of carcinogenesis due to asbestos is 30 to 40 years, and it is negative for Ames test. As described above, it has recently become clear that there are portions of fibrous nanomaterials that cannot be dealt with by conventional toxicological methods.

  Furthermore, as a result of recent research, the cause of malignant mesothelioma is that inhaled asbestos diffuses in the lungs and reaches the deep alveolar region, and penetrates the lungs and pleura in the deep alveolar region deeply. It is thought that the mesothelial cells become malignant. In the future, due to the development of various nanomaterials and the establishment of mass production technology, the use of many nanomaterials including single-layer / multi-layer CNTs is expected to expand, so in addition to conventional animal experiments, Establishment of a new and simple evaluation method for evaluating toxicity and carcinogenicity to humans is desired. In particular, the diffusion status of nanomaterials in the lungs, especially whether they reach the deep part of the alveolar region, and whether the nanomaterial that has reached the deep part of the alveolar region penetrates the lungs / pleura and leaks into the chest cavity Although it is considered that the evaluation is important as one of the indicators for confirming safety, such experimental devices and methods are not known at present.

Atsuya Takagi et al. , “Induction of mesothelioma in p53 +/− mouse by intraperitoneal application of multi-wall carbon nanotube.”, The Journal of the World. 33, no. 1, p105-116 Nagai H et al. , "Diameter and rigidity of multi-walled carbon nanotubes are critical factors in mesogenic injuries and calcinogenes.", Proc Natl. Julie Muller et al. , “Respiratoryity of multi-wall carbon nanotubes”, Toxology and Applied Pharmacology, 207, (2005), p221-231. Jurgen Pauluhn, "Subchronic 13-Week Inhalation Exposure of Rats to Multiwalled Carbon Nanotubes: Toxic Effects Are Determined by Density of Agglomerate Structures Not Fibrillar Structures", TOXICOLOGICAL SCIENCES, 113 (1), p226-242 (2010) Ma-Hock L et al. , “Inhalation toxicity of multiwall carbon nanotubes in rats exposed for 3 months”, Toxicol Sci. 2009 Dec, 112 (2), p468-81

  The present invention has been made in order to solve the above-mentioned conventional problems, and as a result of extensive research, both lungs (hereinafter referred to as “lung etc.”) may be described from the trachea and bronchi taken from the animal. ) In an airtight container, and by repeating negative pressure → atmospheric pressure → negative pressure → atmospheric pressure in the container, lung respiration can be artificially created ex vivo, and the diffusion status of nanomaterials to the lungs, etc. In particular, it has been found that it is possible to quickly evaluate whether or not nanomaterials that reach the alveolar region deeply and whether or not the nanomaterial that has reached the alveolar region deeply penetrates the lung and pleura and leaks into the thoracic cavity. completed.

  That is, an object of the present invention is to provide a nanomaterial diffusion evaluation device, an evaluation method, and a negative pressure generator.

  The present invention relates to a nanomaterial diffusion evaluation apparatus, an evaluation method, and a negative pressure generation apparatus described below.

(1) A nanomaterial diffusion evaluation apparatus including a lung placement container for attaching both lungs from the trachea and bronchus taken out from an animal, and a negative pressure generator for creating a negative pressure inside the lung placement container.
(2) The device according to (1) above, wherein the negative pressure generator is capable of alternately repeating negative pressure and atmospheric pressure in the lung installation container.
(3) The apparatus according to (2) above, wherein the alternating repetition of the negative pressure and the atmospheric pressure is 0.1 to 60 times / min.
(4) The lung installation container is an airtight container, and one end is opened to the atmosphere, and the other end includes a tube to which both lungs are attached from the trachea and bronchus, and a hole connected to a negative pressure generator. The device according to any one of (1) to (3) above.
(5) One end is opened to the atmosphere, and the other end is attached to the other end of the tube located in the lung container with both lungs from the trachea and bronchus removed from the animal. A method for evaluating diffusion of nanomaterials, characterized in that the lungs are artificially breathed by repeating alternately and the diffusion state of the nanomaterials from the trachea / bronchi to both lungs is evaluated.
(6) The method according to (5) above, wherein the negative pressure and the atmospheric pressure are repeated 0.1 to 60 times / minute.
(7) The method according to (5) or (6) above, wherein the negative pressure is 0.01 to 3.0 kPa.
(8) A negative pressure generator capable of alternately repeating negative pressure and atmospheric pressure in an airtight container.
(9) The apparatus according to claim 8, wherein the repetition of the negative pressure and the atmospheric pressure is 0.1 to 60 times / minute.

Since the present invention can reproduce an artificial respiratory system ex vivo using both lungs from the trachea and bronchus taken out of the animal, the nanomaterial trachea and bronchi can be transferred from the trachea and bronchus to both lungs without using a living animal. Can be evaluated.
In addition, since the lung respiration rate and the like can be arbitrarily controlled, it is possible to perform evaluation in a short time in a biomaterial administration of the nanomaterial in a short time.
Furthermore, since the solution in the container in which the lungs are placed can be sampled periodically, it is possible to evaluate changes over time in the nanomaterial that broke through the lung and leaked into the chest cavity, which was difficult to evaluate in vivo. Can do.

FIG. 1 is a schematic diagram illustrating an example of a nanomaterial diffusion evaluation apparatus 10 and a negative pressure generation apparatus 20 according to the present invention. FIG. 2 is a top view of the negative pressure generator 20. FIG. 3 is a drawing-substituting photograph, which is an optical micrograph of the pathological specimen prepared in Example 1. FIG. 4 is an optical micrograph of the pathological specimen prepared in Comparative Example 1 as a drawing-substituting photograph. FIG. 5 is a drawing-substituting photograph, which is an optical micrograph of the pathological specimen prepared in Example 2. FIG. 6 is a drawing-substituting photograph and an optical micrograph of the pathological specimen prepared in Comparative Example 2.

  The nanomaterial diffusion evaluation apparatus, evaluation method, and negative pressure generator of the present invention will be described in detail below.

  FIG. 1 shows an example of a nanomaterial diffusion evaluation device and a negative pressure generator of the present invention. The nanomaterial diffusion evaluation apparatus 10 of the present invention includes at least a negative pressure generator 20 and a lung placement container 30.

  The negative pressure generating device 20 is for making negative pressure in the inside of a lung installation container 30 to be described later. In the example shown in FIG. 1, in the example shown in FIG. 1, the motor 21, the rotating disk 22 that rotates by the driving force of the motor 21, the rotation An adjustment pin 23, a syringe 24, and the adjustment pin that are provided on the plate 22 and can adjust the suction capacity of air in the lung-installed container (pressure in the lung-installed container) by adjusting the mounting position on the turntable 22 23 includes a movable plate 25 having a long hole through which it is inserted and connected to the plunger of the syringe 24, and a guide 26 of the movable plate 25.

  The lung installation container 30 includes a tube 31 and a hole 32, one end of which is open to the atmosphere and the other end to which a lung or the like can be attached. In order to make the inside of the lung installation container 30 into a negative pressure, the other end of the tube 27 or the like, one end of which is connected to the syringe 24 of the negative pressure generating device 20, is connected to the hole 32 using a tube or the like. What is necessary is just to connect the lung installation container 30 and the negative pressure generator 20 airtightly. The tube 31 and the hole 32 may be provided in the main body of the lung installation container 30 or may be provided in a lid that can be hermetically sealed in the opening of the lung installation container 30. Although not shown, a pressure gauge indicating the pressure in the lung installation container 30 may be connected to the lung installation container 30.

  Next, the operation of the negative pressure generator 20 and a nanomaterial diffusion evaluation method using the nanomaterial diffusion evaluation apparatus 10 will be described.

  First, as shown in FIG. 1, a solution 33 for collecting the nanomaterial that broke through the lungs in the lung placement container 30 and keeping the lungs in a wet state is injected into the lung placement container 30. The solution 33 may be a commercially available cell culture solution, physiological saline, or the like. Next, a trachea such as a lung taken out from the animal is attached to the tube 31 in an airtight state using a thread or the like. The nanomaterial may be taken into the lungs by placing the open portion of the tube 31 in an atmosphere in which the nanomaterial to be evaluated is scattered, and the nanomaterial is removed from the opening of the tube 31. Alternatively, the nanomaterial may be administered to the trachea in advance, and then the lungs or the like may be attached to the tube 31.

  The lung is not particularly limited as long as it is a lung extracted from an animal such as a rat, mouse, rabbit, pig, goat or the like, and the size of the lung installation container 30 and the tube 31 depending on the size of the lung to be used. The diameter of the liquid, the injection amount of the solution 33, and the amount of air sucked by the negative pressure generator to be described later may be appropriately adjusted.

  The nanomaterial to be evaluated is not particularly limited as long as it is a nanometer scale (1 nm to 999 nm) material. For example, carbon black, silica, titanium oxide, zinc oxide, fullerene, single-layer / multi-layer CNT, dendrimer, silver / Gold + inorganic fine particles, nanoclay, and the like can be given. The material to be evaluated is not limited to the nanomaterial, and may be a newly developed material in the future. Further, the size is not limited to the nanometer scale, and may be larger than the nanometer scale as long as the influence on the living body can be evaluated using the lung or the like of the present invention.

  FIG. 2 is a top view of the negative pressure generator 20, and the same reference numerals as those in FIG. 1 denote the same members. When the motor 21 rotates, the adjustment pin 23 attached to the turntable 22 rotates, for example, clockwise. Since the adjustment pin 23 is inserted into the long hole 28 provided in the movable plate 25, the movable plate 25 repeats the movement in the left-right direction in FIG. 2 as the adjustment pin 23 rotates. Furthermore, since part of the movable plate 25 is connected to the plunger of the syringe 24, the operation of pulling and pushing the plunger is repeated as the movable plate 25 moves. Since the tip of the syringe 24 and the lung installation container 30 are connected by airtight vinyl, silicon tube or the like, as a result, the pressure in the lung installation container 30 is alternately repeated to negative pressure and atmospheric pressure. be able to. Since the lungs and the like are airtightly attached to the tube 31 in the lung installation container 30, the plunger is pulled and the inside of the lung installation container 30 is inflated, and when the plunger is pushed and returned to the atmospheric pressure, the original pressure is obtained. Return to size. Furthermore, since the other end of the tube 31 to which the lungs or the like are attached is open to the atmosphere, the animal respiratory system can be reproduced ex vivo.

  The ex vivo respiratory system is preferably an environment close to the expansion and contraction of the lungs when a human normally breathes, and the negative pressure in the lung installation container 30 is 0.4 to 0.8 kPa. What is necessary is just to adjust the quantity of the air suck | inhaled, considering the capacity | capacitance of the lung installation container 30, etc. FIG. The “negative pressure” in the present invention means not the absolute value of the pressure but the pressure difference from the atmospheric pressure (about 101.3 kPa). The pressure can be adjusted, for example, by providing a groove in the radial direction of the turntable 22 so that the adjustment pin 23 is slidably engaged and the adjustment pin 23 is fixed using a screw or the like at an arbitrary position, or a plurality of holes are provided in the radial direction. The movable range of the movable plate 25 may be adjusted by providing the adjustment pin 23 in an arbitrary hole. Further, the amount of air to be sucked may be changed by changing the diameter of the syringe 24 instead of changing the position of the adjustment pin 23. Moreover, what is necessary is just to adjust the rotational speed of the motor 21 so that the frequency | count of pushing and pulling of a plunger may be performed about 10-12 times / minute which is the number of lung respirations when a person breathes normally.

  Note that the negative pressure generating device 20 mechanically sucks air to make the inside of the lung installation container 30 have a negative pressure. For example, a certain amount of air is sucked by a vacuum pump, and the lung installation container is sucked after suction. The pressure in the lung installation container 30 may be returned to the atmospheric pressure by opening a valve or the like provided at 30.

  In addition, the above-mentioned pressure and the number of push-pull times are those when reproducing normal human respiration, for example, when conducting a screening of diffusivity of the nanomaterial deep into the alveolar region and the pleura in a short period of time, When evaluating diffusion under mild conditions, it is not necessary to have the above value, and for example, the negative pressure in the lung installation container 30 may be adjusted to be about 0.01 to 3.0 kPa. In addition, the number of times the plunger is pushed and pulled may be appropriately adjusted to about 0.1 to 60 times / minute.

  After administering nanomaterial and breathing artificially for a certain period of time, the lungs etc. are taken out, pathological specimens are created by normal fixation, embedding, slicing, smearing, staining procedures, and microscopic observation is performed. What is necessary is just to evaluate the presence or absence of nanomaterial diffusion. In addition, the nanomaterial leaked into the solution 33 through the lungs and pleura may be observed with a microscope by centrifuging the solution 33 and determining whether or not the nanomaterial is contained in the sediment.

  The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.

<Example 1>
A 12-week-old Brown Norwegian-Fischer hybrid first male rat was thoracotomized and laparotomized under anesthesia, and refluxed with phosphate buffered saline from the right atrium, followed by trachea / bronchi and both lungs Was carefully taken out so as not to be damaged. As the nanomaterial sample, crocidolite (Aoishi cotton), which is available from UICC (Unio Internationalis Contracanum) and has been confirmed to be carcinogenic for mesothelioma by intraperitoneal administration to rats, was used. After administration of 1 mg of crocidolite into the trachea of the removed rat lung, the trachea is immediately and airtightly attached to the tube in the lung container of the present invention using a sterilized blade ESLON suture (USP3-0, Akiyama Seisakusho). It was. About 110 ml (about half of the volume) of Hank's Balanced Salt Solution was placed in a lung placement container having a size of 7.5 (width) × 7.0 (depth) × 4.5 cm (height). Next, the plunger of the syringe of the negative pressure generator is moved at a rate of 15 times per minute with a stroke of about 12 ml, and accordingly the pressure in the lung placement container becomes a negative pressure of about atmospheric pressure to about 2 kPa at maximum, The movement of the lungs during breathing was artificially created and the operation was continued for 5 hours. In addition, the negative pressure of a present Example is about 3 times compared with the human physiological chest negative pressure. After 5 hours, the lungs and the like were carefully collected, fixed with 10% neutral formalin, embedded in paraffin, and pathological specimens were prepared by a conventional method. Hematoxylin and eosin staining and iron staining were performed, and it was evaluated by light microscopy which part of the tissue such as lung was observed with crocidolite. At the same time, the solution in the lung container was collected, centrifuged at 3,500 G for 10 minutes, and the sediment was observed with a microscope to evaluate whether crocidolite leaked out of the lungs.

  FIG. 3 (1) is a low-magnification optical micrograph of the pathological specimen prepared in Example 1, and the arrow above (1) indicates the crocidolite in the bronchi. (2) is an enlarged photograph of the part surrounded by □ in (1), and the arrow in (2) indicates crocidolite that has reached the deep part of the alveolar region and the pleura, which is the outer membrane of the lung.

  Moreover, when the deposit of the solution in the lung installation container of Example 1 was observed with the microscope, crocidolite was confirmed.

<Comparative Example 1>
Except that the negative pressure generator was not operated, an experiment was performed in the same procedure as in Example 1 to create a pathological specimen.

  FIG. 4 (1) is a low-magnification optical micrograph of the pathological specimen prepared in Comparative Example 1, (2) is an enlarged photograph of the part surrounded by □ below (1), and the arrow is crocidolite in the bronchi Is shown. (3) is an enlarged photograph of the part surrounded by □ above (1). As is clear from the photograph, the arrival of crocidolite in the deep part of the alveolar region was not observed.

  Moreover, when the deposit of the solution in the lung installation container of the comparative example 1 was observed with the microscope, the crocidolite was not confirmed.

<Example 2>
An experiment was performed in the same procedure as in Example 1 except that multilayer CNT (diameter 50 nm, average length of about 5 μm, VGCF-S manufactured by Showa Denko KK) was used as the nanomaterial sample instead of crocidolite.

  FIG. 5A is a low-magnification optical micrograph of the pathological specimen prepared in Example 2, and the arrows indicate the multi-walled CNTs in the bronchi. (2) is an enlarged photograph of the part surrounded by □ above (1), and (3) is an enlarged photograph of the part enclosed by □ below (1). The arrows in (2) and (3) indicate multi-walled CNTs that have reached the deep part of the alveolar region and the pleura, which is a film outside the lungs.

  Moreover, when the deposit of the solution in the lung installation container of Example 2 was observed with the microscope, multilayer CNT was confirmed.

<Comparative example 2>
An experiment was performed in the same procedure as in Example 2 except that the negative pressure generator was not operated, and a pathological specimen was prepared.

  FIG. 6 (1) is a low-magnification optical micrograph of the pathological specimen prepared in Comparative Example 2, (2) is an enlarged photograph of the part surrounded by □ above (1), and the arrow is a multilayer in the bronchus CNT is shown. (3) is an enlarged photograph of the part surrounded by □ below (1). As is clear from the photograph, the arrival of multi-walled CNTs in the deep part of the alveolar region was not observed.

  Moreover, when the deposit of the solution in the lung installation container of the comparative example 2 was observed with the microscope, multilayer CNT was not confirmed.

The above Examples 1 and 2 and Comparative Examples 1 and 2 were comprehensively evaluated as follows.
Grade 0: The sample is present only in the bronchi.
Grade 1: The sample reaches the alveoli but stays up to ½ proximal to the pleura.
Grade 2: The sample reaches the distal half of the alveoli but not the pleura.
Grade 3: The sample reaches the pleura but is not detected outside the lungs.
Grade 4: The sample crosses the pleura and is detected in solution outside the lungs.

  Both Example 1 (crocidolite) and Example 2 (multilayer CNT) were grade 4, whereas Comparative Examples 1 and 2 were grade 0. From this result, when the nanomaterial is aspirated by using the evaluation apparatus of the present invention, it is possible to quickly and appropriately evaluate the diffusion to the outside of the lung beyond the alveolar region deep part, the pleura, and the pleura. .

  By using the nanomaterial diffusion evaluation apparatus of the present invention, it is possible to evaluate the diffusion to the deep part of the alveolar region and the pleura in an environment very close to the case where the nanomaterial is sucked into the living body while being ex vivo. Therefore, it is useful as a new evaluation index independent of other evaluations when comprehensively evaluating the safety of nanomaterials at research and development organizations of nanomaterials, manufacturers using nanomaterials, medical institutions, etc. It is.

Claims (9)

  An apparatus for evaluating diffusion of nanomaterials, comprising a lung placement container for attaching both lungs from the trachea and bronchi taken out of an animal, and a negative pressure generator for creating a negative pressure inside the lung placement container.   The device according to claim 1, wherein the negative pressure generating device is capable of alternately repeating negative pressure and atmospheric pressure in the lung-installed container.   The apparatus according to claim 2, wherein the alternating repetition of the negative pressure and the atmospheric pressure is 0.1 to 60 times / minute.   The lung installation container is an airtight container, and one end is opened to the atmosphere, and the other end includes a tube to which both lungs are attached from the trachea and bronchus, and a hole connected to a negative pressure generator. Item 4. The apparatus according to any one of Items 1 to 3.   Attach both lungs from the trachea and bronchus removed from the animal to the other end of the tube located in the lung installation container at one end and open the atmosphere to the other, and negative pressure and atmospheric pressure in the lung installation container alternately A method for evaluating diffusion of nanomaterials, characterized in that the lungs are artificially breathed by repeating the above and the diffusion state of the nanomaterials from the trachea and bronchus to both lungs is evaluated.   The method according to claim 5, wherein the repetition of the negative pressure and the atmospheric pressure is 0.1 to 60 times / minute.   The method according to claim 5 or 6, wherein the negative pressure is 0.01 to 3.0 kPa.   A negative pressure generator capable of alternately repeating negative pressure and atmospheric pressure in an airtight container. The apparatus according to claim 8, wherein the repetition of the negative pressure and the atmospheric pressure is 0.1 to 60 times / minute.