| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | September 17, 2002 |
| PATENT TITLE |
Method for measuring amount of active oxygen produced by leukocytes and oxidative stress |
| PATENT ABSTRACT |
A method for measuring luminol enhancing light emission without separating leukocytes and erythrocytes, that is, using whole blood and a method for measuring the number of leukocytes trapped by the capillary bed or the time required for leukocytes to pass through the capillary bed to thereby provide an effective index for ability of leukocytes to protect infection and for oxidative stress caused by leukocytes are provided. Specifically, a method is provided for measuring an amount of active oxygen produced by leukocytes and oxidative stress, where the method includes allowing an anticoagulated whole blood sample collected from a human or an animal to pass through a microchannel array including a substrate with a fine channel array arranged on its surface and a transparent substrate which is adhered onto the substrate by contact bonding, and measuring a light emission level from whole leukocytes passing through the microchannel array through the transparent substrate to use the measured light emission level as an index of an amount of active oxygen produced by leukocytes and oxidative stress. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | January 24, 2000 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Kikuchi et al. Proc. SPIE-Int. Soc. Opt. Eng., vol. 2978, pp. 165-171, 1997.* Kikuchi. Microvascular Research, vol. 50, pp. 288-300, 1995.* Kikuchi et al. Microvascular Research, vol. 47, pp. 126-139, 1994.* "Standardization of Measurement of Oxidative Stress of Blood Samples by Detection of Luminol Enhancing Chemiluminescence--Aiming at Monitoring Time Course of in Vivo Oxidative Stress," Journal of Japanese Pharmacology, vol. 111, No. 3, 1998, pp. 177-186. (with English Abstract). |
| PATENT CLAIMS |
What is claimed is: 1. A method for measuring an amount of active oxygen produced by leukocytes and oxidative stress, wherein said method comprises allowing an anticoagulated, undiluted whole blood collected from a human or an animal to pass through a microchannel array comprising a channeled substrate with one or more channel arrays arranged on its surface and a transparent substrate bonded to said channeled substrate, and measuring through said transparent substrate a light emission level from whole leukocytes passing through said microchannel array to use the measured light emission level as an index of an amount of active oxygen produced by leukocytes and oxidative stress. 2. The method according to claim 1, wherein said light emission level is measured with or without stopping the flow of the whole blood sample. 3. The method according to claims 1 or 2, wherein the light emission level is measured using a photomultiplier in combination with a direct current amplifier. 4. The method according to claim 3, whereim said method comprises providing a rotating stage on which plural microchannel array holders each of which is capable of holding the microchannel array can be mounted, allowing whole blood samples to flow in plural microchannel arrays in turn, transferring said microchannel arrays onto said rotating stage to measure the light emission level in turn, and measuring the light emission level repeatedly from a microchannel array when the measurement is done for the previous microchannel array on the rotating stage. 5. The method according to claim 3, wherein a substance that amplifies light emission is added to the whole blood sample, and then said whole blood sample is allowed to flow in the microchannel array to measure the light emission level. 6. The method according to claim 3, wherein a leukocyte-stimulating substance, a leukocyte-stimulating cell, or a leukocyte-stimulating particle is added to the whole blood sample, and then said whole blood sample is allowed to flow in the microchannel array to measure the light emission level. 7. The method according to claims 1 or 2, wherein the light emission level is measured using a photomultiplier in combination with a photoelectronic counter. 8. The method according to claim 7, wherein said method comprises providing a rotating stage on which plural microchannel array holders each of which is capable of holding the microchannel array can be mounted, allowing whole blood samples to flow in plural microchannel arrays in turn, transferring said microchannel arrays onto said rotating stage to measure the light emission level in turn, and measuring the light emission level repeatedly from a microchannel array when the measurement is done for the previous microchannel array on the rotating stage. 9. The method according to claim 7, wherein a substance that amplifies light emission is added to the whole blood sample, and then said whole blood sample is allowed to flow in the microchannel array to measure the light emission level. 10. The method according to claim 7, wherein a leukocyte-stimulating substance, a leukocyte-stimulating cell, or a leukocyte-stimulating particle is added to the whole blood sample, and then said whole blood sample is allowed to flow in the microchannel array to measure the light emission level. 11. A method for assessing an effect of a substance selected from the group consisting of drugs, foods, and substances made from ingredients of drugs and ingredients of foods to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress, wherein said method comprises allowing a first sample of anticoagulated, undiluted whole blood collected from a human or an animal to flow in a microchannel array comprising a channeled substrate with one or more channel arrays arranged on its surface and a transparent substrate bonded to said channeled substrate, measuring through said transparent substrate a light emission level from said first sample from whole leukocytes passing through said microchannel array, allowing a second sample of said anticoagulated, undiluted whole blood to which said substance selected from the group has been added to flow in the microchannel array, measuring through said transparent substrate a light emission level from said second sample from whole leukocytes passing through said microchannel array, and comparing the light emission levels from said first sample and said second sample using the measured light emission levels as an index for the effect of the substance selected from the group consisting of drugs, foods, and substances made from ingredients of drugs and ingredients of foods to enhance or suppress an amount of active oxygen produced by the leukocytes of the blood sample and oxidative stress. 12. A method for assessing an effect of a substance selected from the group consisting of drugs, foods, and substances made from ingredients of drugs and ingredients of foods to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress, wherein said method comprises allowing a first sample of anticoagulated, undiluted whole blood collected from a human or an animal to flow in a microchannel array comprising a channeled substrate with one or more channel arrays arranged on its surface and a transparent substrate bonded to said channeled substrate; measuring through said transparent substrate a light emission level from said first sample from whole leukocytes passing through said microchannel array; allowing a second sample of said anticoagulated, undiluted whole blood, collected from said human or said animal after said substance selected from the group has been administered in vivo, to flow in the microchannel array; measuring through said transparent substrate a light emission level from said second sample from whole leukocytes passing through said microchannel array; and comparing the light emission levels from said first sample and said second sample using the measured light emission levels as an index for the effect of the substance selected from the group consisting of drugs, foods, and substances made from ingredients of drugs and ingredients of foods to enhance or suppress an amount of active oxygen produced by the leukocytes of the blood sample and oxidative stress. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to a method for measuring an amount of active oxygen produced by leukocytes and oxidative stress, more specifically to a method for measuring an amount of active oxygen produced by leukocytes and oxidative stress, and a method for assessing an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress. BACKGROUND OF THE INVENTION Production of active oxygen by leukocytes is inevitable for killing bacteria. A high active oxygen-productive capability of leukocytes is very important for protection against infection. Active oxygen is, however, known to damage tissues and DNA. For example, ischemia reperfusion injury is thought to be caused by active oxygen, which would probably be produced by leukocytes. In order to prevent diseases, it is very important to measure an amount of active oxygen produced by leukocytes, and, to develop a method for increasing or decreasing, case by case, the amount of produced active oxygen. Luminol enhancing light emission is widely used at the laboratory level for measurement of an amount of active oxygen produced by leukocytes. This method is performed by measuring faint light emitted from luminol when it is oxidized by various active oxygen species. Several substances other than luminol are also used for enhancement. Here, the term "luminol enhancing light emission" also includes light emission obtained by enhancement by substances other than luminol. This method is simple, but requires to separate leukocytes from erythrocytes since light absorbance by erythrocytes must be prevented. The method for separating these blood cells is complicated and time-consuming. Furthermore, leukocytes largely change their state during the separation procedure, which raises the problem that the results of the measurement are not reliable. To solve this problem, a method of measuring whole blood diluted to about to is proposed (Takayama, Eto, and Yamanaka, Standardization of Measurement of Oxidative Stress of Blood Samples by Detection of Luminol Enhancing Chemiluminescence--aiming at monitoring time course of in vivo oxidative stress--, Journal of Japanese Pharmacology 111(3), 177-186). However, dilution itself is thought to cause changes of the state of leukocytes. Furthermore, the dilution inevitably leads to considerable reduction of enhancement. The conventional methods cannot thus be effective for clinical and diagnostic use due to the above problems. SUMMARY OF THE INVENTION An objective of the present invention is to provide a method for measuring luminol enhancing light emission without separating leukocytes and erythrocytes, that is, using whole blood. This method is able to solve the problems attributable to the procedure for separating leukocytes from erythrocytes, to remarkably increase reliability of the measurement, and to considerably improve the measuring efficiency. Considering protection by leukocytes against infection and damages of tissues, namely oxidative stress, the problems to be solved include not only the amount of active oxygen produced by leukocytes but also (1) how many leukocytes gather at the site of infection or are trapped by the capillary bed at that site, or (2) how long leukocytes take to pass through the capillary bed. In other words, a product of the amount of active oxygen produced by each leukocyte and the above (1) or (2) is considered to be a good index for ability to protect against infection and oxidative stress. However, no effective method has been proposed so far for measuring the number of leukocytes trapped by the capillary bed or the time required for leukocytes to pass through the capillary bed. Another object of the present invention is to provide a method for measuring a product of the amount of active oxygen produced by each leukocyte and the number of leukocytes trapped by the capillary bed or the time required for leukocytes to pass through the capillary bed to thereby provide an effective index for ability of leukocytes to protect against infection and oxidative stress caused by leukocytes. A final object of the present invention is to improve the measurement efficiency so as to provide an effective measuring method which can be applied to clinical and diagnostic use. A first aspect of the invention provides a method for measuring an amount of active oxygen produced by leukocytes and oxidative stress, wherein said method comprises allowing an anticoagulated whole blood sample collected from a human or an animal to flow in a microchannel array comprising a substrate with one or more fine channel arrays arranged on its surface and a transparent substrate which is adhered onto said substrate by contact bonding, and measuring a light emission level from whole leukocytes passing through said microchannel array through said transparent substrate to use the measured light emission level as an index of an amount of active oxygen produced by leukocytes and oxidative stress. A second aspect of the invention provides the method according to the first aspect of the invention, wherein said light emission level is measured with or without stopping the flow of the whole blood. A third aspect of the invention provides the method according to the first or second aspects of the invention, wherein a time-integral value of the measured light emission level is used as an index of an amount of active oxygen produced by leukocytes and of oxidative stress. A fourth aspect of the invention provides the method according to any one of the first to third aspects of the invention, wherein a substance that amplifies light emission is added to the whole blood sample, and said whole blood sample is allowed to flow to measure the light emission level. A fifth aspect of the invention provides the method according to any one of the first to fourth aspects of the invention, wherein a leukocyte-stimulating substance, a leukocyte-stimulating cell, or a leukocyte-stimulating particle is added to the whole blood sample, and said whole blood sample is allowed to flow to measure the light emission level. A sixth aspect of the invention provides the method according to any one of the first to fifth aspects of the invention, wherein the light emission level is measured using a photomultiplier in combination with a direct current amplifier. A seventh aspect of the invention provides the method according to any one of the first to fifth aspects of the invention, wherein the light emission level is measured using a photomultiplier in combination with a photoelectronic counter. An eight aspect of the invention provides the method according to any one of the first to seventh aspects of the invention, wherein said method comprises providing a rotating stage on which plural microchannel array holders each of which is capable of holding the microchannel array can be mounted, allowing whole blood samples to flow in the plural microchannel arrays in turn, transferring said microchannel arrays onto said rotating stage to measure the light emission level in turn, and measuring the light emission level repeatedly from the first microchannel array when the measurement is done for the last microchannel array. A ninth aspect of the invention provides a method for assessing an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress, wherein said method comprises allowing an anticoagulated whole blood sample collected from a human or an animal to flow in a microchannel array, measuring a light emission level from whole leukocytes passing through said microchannel array through said transparent substrate, allowing a whole blood sample to which said substance selected from the group has been added to flow in the array, measuring a light emission level from whole leukocytes passing through said microchannel array through said transparent substrate, and comparing the light emission levels between the whole blood sample with said substance selected from the group and the one without said substance selected from the group using the measured light emission levels as an index for an amount of active oxygen produced by leukocytes and for oxidative stress. A tenth aspect of the invention provides a method for assessing an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress, wherein said method comprises allowing an anticoagulated whole blood sample collected from a human or an animal to flow in a microchannel array, measuring a light emission level from whole leukocytes passing through said microchannel array through said transparent substrate, allowing a whole blood sample collected from a human or an animal to which said substance selected from the group has been administered to flow in the microchannel array, measuring a light emission level from whole leukocytes passing through said microchannel array through said transparent substrate, and comparing the light emission levels between the whole blood sample with said substance selected from the group and the one without said substance selected from the group using the measured light emission levels as an index for an amount of active oxygen produced by leukocytes and for oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows an explanatory sectional view of the structure of the microchannel array that is a model for the capillary bed. FIG. 2 shows an explanatory plane view of the substrate depicted in FIG. 1 from the side of the transparent substrate. FIG. 3 shows the structure of the microchannel array that is a model for the capillary bed and the principle of the measurement of light emission from leukocytes without being affected by light absorbance by erythrocytes. FIG. 4 shows an example of the constitution of the measuring system. FIG. 5 shows a device suitable for the measurement of a number of samples. FIG. 6 is a graph showing an example of time course of the light emission level measured in Example. The X- and Y-axes denote time and the light emission level, respectively. FIG. 7 is a graph showing the comparison of the light emission levels 100 minutes after the commencement of the measurement between a whole blood sample from the subject who was given Kurozu (Japanese traditional vinegar) and a whole blood sample to which Kurozu was added. Explanation of Signs 11, substrate 12, transparent substrate 13, photomultiplier 14, leukocyte 15, erythrocyte 16, fine channel array (microchannel array) 17, small bank 18, terrace part 19, large bank 20, space (inlet and outlet of the microchannel array) 21, microchannel array holder 22, dark box case 23, shutter 24, photomultiplier 25, magnetic shield 26, direct current amplifier 27, recorder 31, rotating stage 32, pulse motor 33, dark box case 34, microchannel array holder 35, photomultiplier 36, pulse motor controller 37, amplifier DETATTIED DESCRIPTION OF THE INVENTION The present invention will be illustrated in detail below. In the method of the present invention, light emission from leukocytes is measured based on luminol enhancing light emission. As described above, the luminol enhancing light emission method is performed by measuring faint light emitted from luminol when it is oxidized by various active oxygen species. Several substances other than luminol are also used for the purpose of enhancement. The term "luminol enhancing light emission" used herein includes all the cases using substances other than luminol. First, the invention described in the first aspect of the invention is described. The first aspect of the invention relates to a method for measuring an amount of active oxygen produced by leukocytes and oxidative stress, and is characterized by allowing an anticoagulated whole blood sample collected from a human or an animal to flow in a microchannel array, and measuring a light emission level from whole leukocytes passing through said microchannel array through the transparent substrate to use the measured light emission level as an index of an amount of active oxygen produced by leukocytes and oxidative stress. The microchannel array used in the first aspect of the invention can be constructed by contact bonding of a transparent substrate onto the surface of a substrate where fine channel array is arranged. Though materials for the substrate are not always particularly limited, silicon single crystal is preferably used since the fine channel array can be easily arranged on it. On the surface of such a substrate made of silicon single crystal, various sizes of channel can be arranged by fine processing technique. The width or sectional area of the channel should be almost equal to the diameter or sectional area of the capillary. For example, if the microchannel array channel is made to have the channel with the width of 7 .mu.m and the depth of 4.5 .mu.m, it can be used as a model of the human capillary bed. Human whole blood is mainly described below as a test sample, but whole blood from an animal such as a dog or a cat can also be used. In the first aspect of the invention, an anticoagulated whole blood sample collected from a human or an animal is first allowed to flow in the microchannel array described above. Next, luminol enhancing light emission from whole leukocytes that are passing through the microchannel array is measured through the transparent substrate. The transparent substrate is thus preferably made of Pyrex.RTM. glass and the like material with excellent transparency. The diameter or sectional area of human leukocytes is larger than the width or sectional area of the microchannel array. The leukocytes thus pass through the microchannel array with largely changing their shapes. More specifically, leukocytes pass through the microchannel array with being strongly pressed on the transparent substrate. Therefore, erythrocytes cannot enter between leukocytes and the transparent substrate, and the light emitted from leukocytes by luminol enhancement to the direction of the transparent substrate is not absorbed by erythrocytes. This allows measuring luminol enhancing light emission from leukocytes through the transparent substrate without separating leukocytes from erythrocytes. The leukocytes trapped by the microchannel array do also not interfere the measurement. The light emission level can be measured using a photomultiplier in combination with a direct current amplifier as described in the sixth aspect of the invention. Alternatively, the measurement can be performed by the combination use of a photomultiplier and a photoelectronic counter as described in the seventh aspect of the invention. The light emission level measured in the whole microchannel array is the product between the total number of leukocytes passed through the microchannel array or those trapped by the microchannel array and the average light emission level from each leukocyte. It is clear that this value is an index for the ability to protect against infection or for the oxidative stress. The production of active oxygen by leukocytes gradually increases after leukocytes have been activated or have recognized stimulus. Having reached the maximal value, it gradually decreases. It is preferable to measure this time course. It usually takes 2 to 3 hours per sample. As described in the eighth aspect of the invention, the time course of multiple samples can be measured by providing a rotating stage on which plural microchannel array holders each of which is capable of holding the microchannel array can be mounted, allowing whole blood samples to flow in the plural microchannel arrays in turn, transferring the microchannel arrays onto the rotating stage to measure the light emission level in turn, and measuring the light emission level repeatedly from the first microchannel array when the measurement is done for the last microchannel array. More specifically, a number of the holders on each of which a microchannel array is set are prepared, multiple samples are measured in turn in a short period of time, and, turning back to the first sample, the measurement is repeatedly performed. This method makes it possible to measure the time course of multiple samples. Such measurement is efficiently performed by constituting the rotating stage on which a number of holders are able to be mounted. Furthermore, the measurement efficiency can be remarkably improved. As described in the ninth aspect of the invention, to assess an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress, an anticoagulated whole blood sample collected from a human or an animal is allowed to flow in the microchannel array, the light emission level from whole leukocytes passing through said microchannel array is measured through the transparent substrate, and a whole blood sample to which said substance has been added is allowed to flow in the array, the light emission level from whole leukocytes passing through the microchannel array is measured through the transparent substrate, and the light emission levels between the whole blood sample with said substance selected from the group and the one without said substance selected from the group are compared using the measured light emission levels as an index for an amount of active oxygen produced by leukocytes and oxidative stress. This method enables assessing an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress by adding said substance to a whole blood sample. Examples of the drugs are a steroid agent and the like. Examples of foods include Kurozu, red wine, and the like. Examples of substances made from ingredients of drugs or foods are acetic acid, ethanol, and the like. As described in the tenth aspect of the invention, an anticoagulated whole blood sample collected from a human or an animal is allowed to flow in the microchannel array, the light emission level from whole leukocytes passing through the microchannel array is measured through the transparent substrate, a whole blood sample derived from a human or an animal to which the substance selected from the group has been administered is allowed to flow in the microchannel array, the light emission level from whole leukocytes passing through the microchannel array is measured through the transparent substrate, and the light emission levels between the whole blood sample with the substance and the one without the substance are compared using the measured light emission levels as an index for an amount of active oxygen produced by leukocytes and for oxidative stress. This method enables assessing an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to suppress an amount of active oxygen produced by leukocytes and oxidative stress by administering the substance to a human. Examples of drugs, foods, and substances made from their ingredients are as described above. According to the invention described in the first aspect of the invention, faint light emission from leukocytes can be measured using a whole blood sample without separating leukocytes from erythrocytes. This invention solves the problems attributed to a procedure for separating leukocytes from erythrocytes, remarkably increases reliability of the measurement, and considerably improves the measuring efficiency. The invention described in the first aspect of the invention enables measuring the number of leukocytes trapped by the capillary bed or the time required for leukocytes to pass through the capillary bed, and thus provides a useful index for the ability to protect against infection and for oxidative stress. Since the light emission from the whole microchannel array is measured in accordance with the invention described in the first aspect of the invention, the total light emission level is increased and can be measured using a direct current amplifier at real time. Therefore, the invention is advantageous in that the measurement can be performed when a whole blood sample is allowed to be flowing in the microchannel array without stopping the flow of the sample. For this reason, when the measurement is performed using a photoelectronic counter with stopping the flow of a whole blood sample, the measuring enhancement increases, and the light emission from leukocytes can be measured without adding a leukocyte-stimulating substance, a leukocyte-stimulating cell, or a leukocyte-stimulating particle. In this case where a photoelectronic counter is used, the light emission from leukocytes can be measured advantageously without adding a sensitizer or an enhancer but by prolonging the counting time. According to the invention described in the eighth aspect of the invention, it is possible to measure the time course of a number of samples, thereby remarkably increasing the measuring efficiency. According to the invention described in the ninth aspect of the invention, an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress can be assessed. The invention described in the tenth aspect of the invention also enables measuring an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress. Therefore, the methods of the present invention can be useful for clinical or diagnostic use. If the measurement efficiency is further improved, the methods of the present invention would be extremely effective for clinical and diagnostic use. The present invention will be demonstrated below with reference to Example depicted in Figures, but is not construed to be limited thereto. |
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