Patent Application
20120094321
The present invention relates to a method for measuring a dehydrogenase or a substrate for the dehydrogenase in a sample, and a kit used for the method.
In samples such as blood serum and blood plasma, dehydrogenases or substrates for the dehydrogenases are present at high concentrations. In clinical chemical diagnoses, it is general to use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a coenzyme for the measurement of those enzymes and substrates. Some known examples of a combination of a dehydrogenase and a substrate that are used in such a method, include a lactate dehydrogenase and lactic acid, a glutamate dehydrogenase and glutamic acid, an alcohol dehydrogenase and an alcohol, a glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate, and a glucose-6-phosphate dehydrogenase and glucose-6-phosphate. In this case, in order to measure any one of the members of the combinations, the other member is used as a reagent together with a coenzyme. Similarly, in order to measure a particular substance such as urea in a sample, a technique of subjecting the particular substance to an enzymatic reaction using a reagent so as to produce a particular dehydrogenase or a substrate for the dehydrogenase as an intermediate or a final product, thereby allowing the dehydrogenase or the substrate for the dehydrogenase to react with the coenzyme described above, which results in measuring the particular substance, is also widely performed.
In regard to these measurement methods, it is general to employ a technique in which NAD or NADP is finally converted to a reduced form such as NADH or NADPH by a dehydrogenase reaction, and a dehydrogenase or a substrate for the dehydrogenase is measured based on an increase in the absorbance in an ultraviolet wavelength region (usually, 340 nm), which occurs as a result of such conversion. However, in this case, since it is measured in an ultraviolet region, convenient glass or plastic cannot be used for a measurement cell, and it is necessary to use expensive quartz. In addition, it is also necessary to use an ultraviolet light source and a detector in an analytical apparatus, and therefore, the analytical apparatus has been liable to have a large size and be expensive. As a result, the technique cannot be applied to apparatuses that use a visible spectrophotometer, with which measurement can be made even in the field of dry chemistry or at small hospitals. Therefore, in order to enable the technique to be used in such occasions, there is a demand for a method of measuring a dehydrogenase or a substrate for the dehydrogenase as described above in the visible region.
On the other hand, Patent Literature 1 suggests a method of performing an enzymatic cycling reaction by using the combination of thio-NAD or the thio-NADP and NADH or NADPH as a coenzyme when glucose-6-phosphate is allowed to react with glucose-6-phosphate dehydrogenase, and measuring glucose-6-phosphate using thio-NADH or thio-NADPH, which are both reduced form enzymes produced by the enzymatic cycling reaction, based on the absorbance obtained in the visible region near 400 nm. However, this method uses an enzymatic cycling reaction, and cannot be necessarily said to be a convenient measurement method. Furthermore, the method cannot be said to be a measurement method suitable for measuring objects of measurement at high concentrations.
It is an object of the present invention to provide a method which enables measurement based on the absorbance obtained in the visible region, can be applied to apparatuses that use a visible spectrophotometer and can make measurements even in the field of dry chemistry or at small hospitals, and is capable of accurately measuring a dehydrogenase or a substrate for the dehydrogenase, that is present in a sample at a high concentration.
The present inventors paid attention to the fact that, for example, as described in Patent Literature 1, thio-NAD or thio-NADP, which are both coenzymes used to measure a dehydrogenase or a substrate for the dehydrogenase at a low concentration by an enzymatic cycling method, are able to alter the absorbance in the visible region through an enzymatic reaction, and thus the inventors conducted an investigation on a method for measuring an object of measurement that is present in a sample at a high concentration, using the coenzymes. As a result, it was found that when thio-NAD or thio-NADP is used as a coenzyme to measure an object of measurement at a high concentration, linearity is not easily maintained in a high concentration region, and in addition, the blank value may also be high, so that the object of measurement cannot be measured accurately.
The inventors made an attempt to measure a dehydrogenase or a substrate for the dehydrogenase in a sample by subjecting a certain kind of substance in the sample to an enzymatic reaction using NAD as a coenzyme so as to remove the substance using a certain enzyme, subsequently allowing a dehydrogenase, a substrate for the dehydrogenase and thio-NAD to react using the sample from which the substance has been removed, and then measuring the absorbance in the visible region. However, the inventors surprisingly discovered that when the absorbance in the visible region is measured using, as the sample, a simple constitution in which a dehydrogenase, a substrate for the dehydrogenase, thio-NAD and NAD are co-present, irrespective of the order of addition of NAD and thio-NAD, the reaction blank value decreases, and the gradient of the calibration curve is made low, so that the dehydrogenase or the substrate for the dehydrogenase in the sample can be accurately measured. As a result, it was found that when such a constitution is employed, the problems described above are addressed, and a dehydrogenase or a substrate for the dehydrogenase can be accurately measured even in a high concentration region.
Therefore, in regard to a method for measuring a dehydrogenase or a substrate for the dehydrogenase contained in a sample using the absorbance in the visible region, when thio-NAD or thio-NADP as well as NAD or NADP are both used as coenzymes, the object of measurement can maintain linearity even in a high concentration region, and meanwhile, the blank value of the calibration curve can be kept low. As a result, the inventors found that there can be provided a method for measuring a dehydrogenase or a substrate for the dehydrogenase simply and accurately in a broad region of the concentration of an object of measurement, using a convenient visible spectrophotometer, and thereby completed the present invention.
Therefore, the present invention relates to a measurement method of performing a reaction using a dehydrogenase, a substrate for the dehydrogenase, and coenzymes, and thereby measuring the dehydrogenase or the substrate for the dehydrogenase in a sample, in which the dehydrogenase or the substrate for the dehydrogenase in the sample is measured by performing the reaction using thio-NAD or thio-NADP and NAD or NADP as the coenzymes, and measuring an increase in the absorbance caused by the thio-NADH or thio-NADPH produced from the reaction.
Furthermore, the present invention relates to a measurement method in which a particular substance in a sample is used in an enzymatic reaction to produce a dehydrogenase or a substrate for the dehydrogenase as an intermediate or final product, the dehydrogenase or the substrate for the dehydrogenase is subjected to the measurement method described above, and the particular substance in the sample is measured based on the amount of the dehydrogenase or the substrate for the dehydrogenase that has been produced as an intermediate or a final product.
The present invention also relates to a measurement kit for measuring a dehydrogenase or a substrate for the dehydrogenase, the measurement kit containing, as an essential component, a component which is other than the object of measurement selected between the dehydrogenase and the substrate for the dehydrogenase, and also containing, as an essential components, thio-NAD or thio-NADP and NAD or NADP as coenzymes.
Furthermore, the present invention relates to a measurement kit for measuring a particular substance, the measurement kit containing, as essential components, a component for deriving a dehydrogenase or a substrate for the dehydrogenase from the particular substance through an enzymatic reaction, and a component that is not derived selected between the dehydrogenase and the substrate for the dehydrogenase, and also containing, as essential components, thio-NAD or thio-NADP and NAD or NADP as coenzymes.
According to the present invention, even though a dehydrogenase or a substrate for the dehydrogenase contained in a sample is measured based on the absorbance obtainable in the visible region, the object of measurement can maintain linearity even in a high concentration region by using both thio-NAD or thio-NADP and NAD or NADP as coenzymes, and the blank value of the calibration curve can be kept low. As a result, measurement can be made simply and accurately in a broad region of the concentration of the object of measurement, using a convenient visible spectrophotometer.
As the sample used in the present invention, a biological sample such as blood serum, blood plasma or urine, or a model sample thereof is preferred.
The object of measurement of the present invention is a dehydrogenase or a substrate for the dehydrogenase in a sample, or is a particular substance in a sample, which is capable of producing a dehydrogenase or a substrate for the dehydrogenase as an intermediate or a final product through an enzymatic reaction.
According to the present invention, when a dehydrogenase enzyme reaction is performed, the reaction is carried out by allowing thio-NAD or thio-NADP as well as NAD or NADP to be present as coenzymes. Any of combinations of thio-NAD or thio-NADP and NAD or NADP may be used, but a combination of thio-NAD and NAD, or a combination of thio-NADP and NADP is preferred.
Hereinafter, thio-NAD or thio-NADP may be abbreviated to thio-NAD(P). Also, NAD or NADP may be abbreviated to NAD(P).
According to the present invention, thio-NAD means thionicotinamide adenine dinucleotide, and thio-NADP means thionicotinamide adenine dinucleotide phosphate. Furthermore, NAD means nicotinamide adenine dinucleotide, and NADP means nicotinamide adenine dinucleotide phosphate.
According to the present specification, thio-NADH, thio-NADPH, NADH and NADPH mean reduced forms of the coenzymes, that is, the reduced forms of thio-NAD, thio-NADP, NAD and NADP, respectively. Hereinafter, thio-NADH or thio-NADPH may be abbreviated to thio-NAD(P)H. Furthermore, NADH or NADPH may be abbreviated to NAD(P)H.
According to the present invention, there are no particular limitations on the amount of NAD(P) used, but the amount is preferably 0.5-fold moles or more, more preferably 1.5- to 30-fold moles, and most preferably 3.0- to 20-fold moles, relative to the amount of thio-NAD(P) used. If the amount of NAD(P) is too small, it may be difficult to maintain the linearity of the calibration curve, and the decrease in the blank value may be insufficient. On the other hand, if the amount of NAD(P) is too large, the gradient of the straight line of the calibration curve may be small, and it may be impossible to measure the object of measurement accurately.
In the present invention, a dehydrogenase reaction is carried out using both thio-NAD(P) and NAD(P) as coenzymes, and an increase in the absorbance caused by the thio-NAD(P)H produced from the reaction is measured. The wavelength for the measurement of absorbance is preferably in the range of 395 to 415 nm, and since the measurement can be made in the visible region, the measurement can be made using a visible spectrophotometer that is simple and easy to operate, as the analytic apparatus to be used.
According to the present invention, there are no particular limitations on the dehydrogenase or the substrate for the dehydrogenase, as long as the dehydrogenase or the substrate for the dehydrogenase is, for example, a dehydrogenase or a substrate for the dehydrogenase which can be measured by a dehydrogenase enzyme reaction using NAD(P) or thio-NAD(P) as a coenzyme that is currently used as an object of measurement in the industry of clinical laboratory tests. In this case, the dehydrogenase or the substrate for the dehydrogenase may be a dehydrogenase or a substrate for the dehydrogenase that is initially present in the sample. Further, the dehydrogenase or the substrate for the dehydrogenase may also be a dehydrogenase or a substrate for the dehydrogenase that has been produced as an intermediate or a final product when a particular substance is subjected to an enzymatic reaction in order to measure the particular substance in a sample. In this case, consequently, the particular substance can be measured, depending on the dehydrogenase or the substrate for the dehydrogenase produced from the enzymatic reaction.
Examples of a combination of a dehydrogenase and a substrate that may be used in such a method include a lactate dehydrogenase and lactic acid, a glutamate dehydrogenase and glutamic acid, an alcohol dehydrogenase and an alcohol, a glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate, and a glucose-6-phosphate dehydrogenase and glucose-6-phosphate. In this case, in order to measure any one of the members of such a combination, the other member can be used as a reagent for carrying out a dehydrogenase enzyme reaction, together with coenzymes. According to the present invention, an object of measurement that is present in a sample at a high concentration is suitable.
In the case of using, as the dehydrogenase or the substrate for the dehydrogenase, a dehydrogenase or a substrate for the dehydrogenase that has been produced as an intermediate or a final product when a particular substance in a sample is subjected to an enzymatic reaction so as to measure the particular substance in the sample, an example of using a glucose-6-phosphate dehydrogenase as the dehydrogenase, and glucose-6-phosphate as the substrate for the dehydrogenase. In that occasion, for example, measurement can be made using urea (conventionally, urea may also be determined in terms of urea nitrogen) as a particular substance. In this case, for example, when urea in a sample is subjected to the action of ATP, magnesium ion, potassium ion, hydrogen carbonate ion, and urea amidolyase, which are provided by a reagent, in a first enzymatic reaction to produce ADP; subsequently, as a second enzymatic reaction, this ADP is subjected to the action of a hexokinase derived from a reagent to produce glucose-6-phosphate; and then the glucose-6-phosphate (substrate for the dehydrogenase) thus produced is subjected to the action of a glucose-6-phosphate dehydrogenase (dehydrogenase) as well as thio-NAD(P) and NAD(P) to induce a dehydrogenase enzyme reaction, glucose-6-phosphate (substrate for the dehydrogenase) thus produced can be measured from an increase in the absorbance, which is dependent on the amount of thio-NAD(P)H produced from the reaction. As a result, based on this procedure, urea can be measured as the particular substance in the sample. At this time, an ADP-dependent hexokinase is preferred as the hexokinase.
This example can be represented by the following reaction schemes.
According to the present invention, when a dehydrogenase or a substrate for the dehydrogenase is measured as the object of measurement, for example, a composition containing, as an essential component, a component other than the object of measurement selected between the dehydrogenase and the substrate for the dehydrogenase, and additionally containing, if necessary, other components such as a buffering agent and a surfactant, can be used as a first reagent, and a composition containing thio-NAD(P) and NAD(P) as essential components, and additionally containing other components such as a buffering agent and a surfactant can be used as a second reagent.
In this case, when glucose-6-phosphate is measured as a specific object of measurement, for example, the first reagent contains, as an essential component, glucose-6-phosphate dehydrogenase as the dehydrogenase, and the second reagent contains thio-NAD(P) and NAD(P) as essential components. In addition to these, the first reagent and the second reagent can contain other components such as a buffering agent and a surfactant.
When a dehydrogenase or a substrate for the dehydrogenase is measured as the object of measurement using such a first reagent and such a second reagent, for example, a sample and the first reagent are mixed, and the mixture is left to stand for 0 to 15 minutes. Subsequently, the resulting liquid mixture and the second reagent are mixed, and thereby, an enzymatic reaction is carried out for 0.5 to 15 minutes. The absorbance of the reaction liquid thus obtained is measured at 405 nm with a visible spectrophotometer. When the measured value is compared with a calibration curve that has been prepared in advance, the concentration of the object of measurement in the sample can be measured. Furthermore, in regard to the mixing of the sample and the reagents in this case, first, the first reagent and the second reagent are mixed to prepare a single measuring reagent, and the measuring reagent and the sample can be mixed. Furthermore, the enzymatic reaction can be carried out also by subjecting the resulting mixture to react for 0.5 to 15 minutes.
According to the present invention, when a particular substance is measured as the object of measurement, for example, the first reagent can be allowed to contain, as an essential component, a component for deriving a dehydrogenase or a substrate for the dehydrogenase from the particular substance by an enzymatic reaction, and a component that is not derived selected between the dehydrogenase and the substrate for the dehydrogenase, and can be allowed to additionally contain other components such as a buffering agent and a surfactant, if necessary. The second reagent can be allowed to contain thio-NAD(P) and NAD(P) as essential components, and to additionally contain other components such as a buffering agent and a surfactant.
In this case, when urea as the particular substance is measured as a specific object of measurement, for example, a composition containing first enzymatic reaction-inducing components for producing ADP from urea (that is, urea amidolyase, ATP, Mg2+, K+, and HCO3−), and second enzymatic reaction-inducing components for producing glucose-6-phosphate (that is, substrate for the dehydrogenase) from ADP (that is, glucose and hexokinase), as components that derive glucose-6-phosphate (that is, substrate for the dehydrogenase) from urea (that is, particular substance) through an enzymatic reaction; and glucose-6-phosphate dehydrogenase as a dehydrogenase for the reagent components, can be used as the first reagent. The second reagent contains both thio-NAD(P) and NAD(P) as essential components. In addition to those, the first reagent and the second reagent can contain other components such as a buffering agent and a surfactant.
When the particular substance is measured as the object of measurement using such a first reagent and such a second reagent, for example, the sample and the first reagent are mixed, and the mixture is left to stand for 0 to 15 minutes. Subsequently, the resulting liquid mixture and the second reagent are mixed, and thereby, an enzymatic reaction is carried out for 0.5 to 15 minutes. The absorbance of the reaction liquid thus obtained is measured at 405 nm using a visible spectrophotometer. When the measured value is compared with a calibration curve that has been prepared in advance, the concentration of the object of measurement in the sample can be measured. Furthermore, in regard to the mixing of the sample and the reagents in this case, first, the first reagent and the second reagent are mixed to prepare a single measuring reagent, and the measuring reagent and the sample can be mixed. Furthermore, the enzymatic reaction can be carried out also by subjecting the resulting mixture to react for 0.5 to 15 minutes.
As is obvious from the descriptions given above, according to the present invention, use can be made of a measurement kit for measuring a dehydrogenase or a substrate for the dehydrogenase, which contains, as an essential component, a component other than the object of measurement selected between the dehydrogenase and the substrate for the dehydrogenase, and contains, as essential components, thio-NAD or thio-NADP and NAD or NADP as coenzymes.
In this case, for example, in order to measure a dehydrogenase or a substrate for the dehydrogenase, use can be made of a measurement kit for measuring a dehydrogenase or a substrate for the dehydrogenase, which includes a first reagent containing, as an essential component, a component other than the object of measurement selected between the dehydrogenase and the substrate for the dehydrogenase, and a second reagent containing thio-NAD or thio-NADP and NAD or NADP as essential components.
Furthermore, according to the present invention, in order to measure a particular substance, use can be made of a measurement kit for measuring a particular substance, which contains, as essential components, a component for deriving a dehydrogenase or a substrate for the dehydrogenase from the particular substance through an enzymatic reaction, and a component that is not derived selected between the dehydrogenase and the substrate for the dehydrogenase, and also contains, as essential components, thio-NAD or thio-NADP and NAD or NADP as coenzymes.
In this case, use can be made of, for example, a measurement kit for measuring a particular substance, which includes a first reagent containing, as essential components, a component for deriving a dehydrogenase or a substrate for the dehydrogenase from the particular substance through an enzymatic reaction, and a component that is not derived selected between the dehydrogenase and the substrate for the dehydrogenase, and a second reagent containing thio-NAD or thio-NADP and NAD or NADP as essential components.
In the first and second reagents in these measurement kits, other components such as a buffering agent and a surfactant can be optionally incorporated, as described above.
Hereinafter, the present invention will be described in more detail by way of Examples and a Comparative Example, but the present invention is not intended to be limited to these examples.
In order to measure urea nitrogen as the particular substance in a sample, as a first enzymatic reaction, urea nitrogen in the sample was subjected to the action of ATP, magnesium ion, potassium ion, hydrogen carbonate ion, and urea amidolyase, which were provided by a reagent, to produce ADP. Subsequently, as a second enzymatic reaction, this ADP was subjected to the action of a hexokinase from a reagent to produce glucose-6-phosphate. Subsequently, the glucose-6-phosphate (substrate for the dehydrogenase) thus produced was subjected to the action of glucose-6-phosphate dehydrogenase (dehydrogenase) as well as thio-NAD and NAD, and glucose-6-phosphate (substrate for the dehydrogenase) thus produced was measured from an increase in the absorbance, which is dependent on the amount of thio-NADH produced. Thereby, the urea nitrogen as the particular substance in a sample was measured. Furthermore, as a comparison control, the same measurement was carried out using thio-NAD alone.
In regard to the urea nitrogen measurement systems using urea amidolyase, a hexokinase and glucose-6-phosphate dehydrogenase, measurement systems in which thio-NAD and NAD were mixed as coenzymes (Examples 1, 2 and 3), and a measurement system in which thio-NAD was used alone (Comparative Example 1) were compared. In this case, the urea was quantified as the amount of urea nitrogen. The sample and the reagents were described below.
A urea solution was prepared to a concentration of 75 mg/dL in terms of urea nitrogen, and the urea solution was appropriately diluted with physiological saline to various concentrations.
In the first reagent, the amount of each component was adjusted so as to have the following concentration.
In the second reagent, the amounts of Tris and the surfactant were adjusted so as to have the following respective concentrations.
The addition amounts of thio-NAD and NAD in the second reagent are presented in Table 1. Furthermore, in Comparative Example 1, NAD was excluded from the composition of the above second reagent composition, and thio-NAD was used alone.
The measurement was carried out as described below. 3.0 μL of the sample and 100 μL of the first reagent were mixed for 5 minutes at 37° C. using a visible region analyzer. Subsequently, 100 μL of the second reagent was added to the mixture, and the resulting mixture was allowed to react for 5 minutes at the same temperature. The absorbance after the color development reaction was measured at a principal wavelength of 405 nm and a sub-wavelength of 505 nm, by a one-point end method for the relevant analyzer model.
The results thus obtained are presented in
On the other hand, in the composition which used thio-NAD alone as Comparative Example 1, the linearity exceeded beyond the detectable absorbance range at a urea nitrogen concentration of 25 mg/dL or higher, and therefore, measurement could not be achieved.
According to the present invention, when a dehydrogenase or a substrate for the dehydrogenase contained in a sample is measured by means of an enzymatic reaction, the dehydrogenase or the substrate for the dehydrogenase can be measured based on the absorbance in the visible region, by using thio-NAD or thio-NADP as well as NAD or NADP as coenzymes. Thereby, linearity can be maintained even in a high concentration region of the object of measurement, and the blank value of the calibration curve can also be kept low. As a result, measurement can be made simply and accurately in a broad region of the concentration of the object of measurement, using a convenient visible spectrophotometer.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-175379 | Jul 2009 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2010/060450 | 6/21/2010 | WO | 00 | 12/21/2011 |
