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  Pharmaceutical Patents  

 

Title:  Method of detecting mild impaired glucose tolerance or insulin secretory defect
United States Patent: 
7,452,687
Issued: 
November 18, 2008

Inventors:
 Yamakoshi; Masaru (Mishima, JP), Kouzuma; Takuji (Mishima, JP)
Assignee:
  Asahi Kasei Pharma Corporation (Tokyo, JP)
Appl. No.:
 10/509,120
Filed:
 March 27, 2003
PCT Filed:
 March 27, 2003
PCT No.:
 PCT/JP03/03771
371(c)(1),(2),(4) Date:
 November 29, 2004
PCT Pub. No.:
 WO03/083133
PCT Pub. Date:
 October 09, 2003


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

It is intended to provide a noninvasive method of conveniently detecting mild impaired glucose tolerance and/or insulin hyposecretion at the early stage with the use of an enzyme. Namely, mild impaired glucose tolerance and/or hyposecretion at the early stage are detected by quantifying myoinositol secreted into the urine before loading glucose and after loading glucose for a definite period of time with the use of a reagent and comparing the increase (or the increase ratio) in the myoinositol content thus measured with a characteristic level which has been preliminarily determined in normal subjects.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention intends to provide an assay method for simple determination of mild impaired glucose tolerance and/or insulin secretory defect with good reproducibility.

For achieving this object, the present inventors considered that search for any marker for effectively determining mild impaired glucose tolerance and/or insulin secretory defect was advantageous. As a result of concentrated efforts, whereas myo-inositol is conventionally considered to be useful for detection of insulin resistance and prediabetes (borderline type and diabetes mellitus), the present inventors unexpectedly found that myo-inositol is also useful as a marker for effectively detecting mild impaired glucose tolerance or insulin secretory defect

Blood serum, plasma, or urine collected from the human body, or a homogenized extract of living tissue are used as a sample. Urine is preferable because it can be non-invasively obtained.

The present inventors continued to develop a high-sensitive quantitative determination assay of myo-inositol and a composition for the assay to provide a simple and cost-effective quantitative determination assay of myo-inositol with a high degree of accuracy (JP 06-61278 B). This enzymatic assay, which does not require any preliminary treatment, opened the way to obtain reliable data of myo-inositol for the first time. Such development of the high-sensitive quantitative determination assay and the composition for the quantitative determination allowed the first success of providing the method of the present invention for examining mild impaired glucose tolerance and/or insulin secretory defect.

Furthermore, after the administration of a given amount of glucose to a subject, urine samples were obtained non-invasively from the subject within a given time period and the myo-inositol levels thereof were determined using the myo-inositol assay reagent as described above. The determination revealed that not only individuals with prediabetes (of borderline type, IFG, IGT) and individuals of diabetes mellitus, but also individuals practically showing mild impaired glucose tolerance in spite of being of NGT and individuals practically showing a decrease in early insulin secretion in spite of being of NGT have higher levels than the characteristic value predetermined from healthy individuals. Therefore, it has been found that the assay reagent of the present invention enables not only the distinction between NGT and non-NGT with progressed impaired glucose tolerance (borderline type, IFG IGT, diabetes) but also the simple, highly reproducible and efficient distinction of individuals practically showing mild impaired glucose tolerance in spite of being of NGT or individuals practically showing a decrease in early insulin secretion in spite of being of NGT from healthy individuals.

In addition, the concentration of myo-inositol in a sample may be very low and some of myo-inositol dehydrogenases used may react weakly with glucose. Thus, the elimination of glucose may be required in advance. A method for the elimination of glucose includes one using extreme chemical stability of myo-inositol and one by modifying glucose using an enzyme as a catalyst. The method using the chemical stability includes, for example, one by heating a sample in the presence of 6 N HCl to allow the acid decomposition of sugars except myo-inositol and recovering myo-inositol remained in the decomposed product; and one by treating a sample with a reducing agent such as sodium borohydride to reduce sugars having carbonyl groups or formyl groups such as glucose except myo-inositol and modifying them to make compounds unreactive with myo-inositol dehydrogenase, i.e. an enzyme for the quantitative myo-inositol assay. The method by modifying glucose using an enzyme as a catalyst includes one by converting glucose in a sample into gluconic acid with glucose oxidase (EC1,1,3,4) and one by converting glucose in a sample into glucose-6-phosphate with hexokinase (EC2,7,1,1).

Various improvements are known in these converting methods. In relation to the method of converting glucose into gluconic acid with glucose oxidase, for example, known is a method to eliminate hydrogen peroxide products by catalase after the reaction with glucose oxidase (JP 63-185397 A).

Furthermore, in relation to the method of converting glucose into glucose-6-phosphate with hexokinase, there are known methods to convert glucose into fructose-1,6-bisphosphate using phosphohexose isomerase and 6-phosphofructokinase to prevent glucose-6-phosphate from being reconverted into glucose through an equilibrium reaction (JP 05-76397 A); to perform the reaction with glucose-6-phosphate dehydrogenase in the presence of an oxidized coenzyme (JP 01-320998 A, JP 03-27299 A); and to perform the reaction with pyruvate kinase in the presence of adenosine diphosphate to prevent the change of adenosine triphosphate level decreasing as glucose is eliminated and thus keep the adenosine triphosphate level constant (JP 02-104298 A).

However, when glucose is eliminated using hexokinase, the enzymatic reaction produces a large amount of ADP in the reaction solution, so that the effect thereof on the enzymatic reaction cannot be ignored. Thus, it is preferable to convert the resulting ADP to a compound that does not affect the reaction.

Thus, the present inventors have considered an effective method using an ADP eliminating agent to convert ADP generated in the reaction solution by the enzymatic reaction to a compound that does not affect the reaction.

Any substances capable of converting ADP to a compound that does not affect the reaction can be used as an ADP eliminating agent Of these, enzymes are preferable and kinases that catalyze conversion of ADP to AMP are more preferable. Kinases are also called phosphokinases or phosphotransferases. Examples of the kinases which catalyze conversion of ADP to AMP include pyrophosphate-glycerol transferase, 6phosphofructokinase, acetate kinase, and ADP-hexokinase.

As a result of keen investigations, the present inventors have found that 6phosophofructokinase and ADP-hexokinase are preferably used as an ADP eliminating agent in the present invention.

When 6phosphofructokinase is used as an ADP eliminating agent in the reaction of eliminating glucose in a sample, ADP is produced along with the conversion of glucose to glucose-6-phosphate using ATP-hexokinase in the presence of ATP and is simultaneously reacted with 6-phosphofructokinase to allow the conversion of ADP to AMP along with the conversion of preadded fructose-6-phosphate to fructose-1,6-bisphosphate.

When ADP-hexokinase is used as an ADP eliminating agent in the reaction of eliminating glucose in a sample, ADP is produced along with the conversion of glucose to glucose-6phosphate using ATP-hexokinase in the presence of ATP and can be converted to AMP.

In addition, it is preferable to perform such reaction in the presence of salts. Examples of the salts include: magnesium salts such as magnesium chloride and magnesium acetate; and potassium salts such as potassium chloride and potassium sulfate. The concentration of salts used is, but not limited to, preferably about 1 to 100 mM.

Any of these compounds produced by the enzymatic modification are not reactive with myo-inositol dehydrogenase, an enzyme for the quantitative myo-inositol determination. The present inventors have found that it is more preferable to previously eliminate glucose by these methods.

Further, the present inventors have found that myo-inositol can be determined more accurately when two kinds of kinases, ATP-hexokinase and ADP-hexokinase, are used simultaneously because the influence of sugars in a sample is reduced. In addition, the present inventors have found that the range of myo-inositol determination can be extended by about 10 times by adjusting thio-NAD level to a final concentration of 0.1 mM or more, preferably 2 to 10 mM. Thus, the present inventors have completed a higher sensitive assay system.

The term "characteristic value" refers to a value determined on the basis of an average of myo-inositol levels in urine samples of healthy subjects selected from those of NGT; standard deviation; and ROC (response operating characteristic) curve. When urine samples are used, the increment in urinary myo-inositol excretion between before the glucose load and at a predetermined time after the glucose load is in the range of 0 to 20 .mu.g/mg creatinine; or 5 to 15 .mu.g/mg creatinine; or more preferably 8 to 12 .mu.g/mg creatinine. In addition, the characteristic value may be changed if a large-scale examination is conducted in the future and the determination is conducted for healthy individuals selected clinically. Furthermore, the characteristic value may also vary depending on the selected populations of race, sex, and age.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, the detection of mild impaired glucose tolerance and insulin secretory defect is carried out by determining the amounts of myo-inositol excreted in urine of a subject before the glucose load and at a predetermined time after the glucose load using the reagent of the present invention; and making a comparison of an increasing amount or increasing rate of myo-inositol between before and after the glucose load with the characteristic value defined in advance for healthy individuals.

The increasing amount is calculated as a difference between the myo-inositol content at a predetermined time after the glucose load and the myo-inositol content before the glucose load, and the increasing rate is calculated as a ratio of the myo-inositol content at a predetermined time after the glucose load to the myo-inositol content before the glucose load.

For the concentration of myo-inositol, an actually determined value may be used, or a relative value with respect to an appropriate standard index may be used for compensating the dilution of urine with drinking water. Preferably, the index is a urinary creatinine level. The subjects include all individuals in addition to those suspected of being lifestyle-related diseases such as diabetes.

Any amounts of glucose loaded and any types of the glucose loading methods may be used. However, preferable is an oral administration of an aqueous 75 g glucose solution as used in a typical glucose load test or a meal ingestion.

Urine samples may be collected before the glucose load and at any times until 6 hours passed just after the glucose load, preferably at 30 minutes to 3 hours after the glucose load. A urine collection period is suitably selected from 30 minutes to 3 hours.

When using urine as a sample, it is collected by a non-invasive method, so that there is no need to select the sampling method, time, and place. For instance, such a sample can be easily prepared by the subject at home, office, school, or the like, and the collected urine sample may be transported directly or in a form of urine-immersed filter paper or other suitable forms, these eliminating the need to be tied to medical institutions or the like. Thus, the present invention provides a prominent method in which, when a filter paper or the like is impregnated with urine, the sample dispatched is extracted by a suitable method and provided to the simple and rapid assay of the present invention and then immediately the results is sent to the subject.

In particular, urinary myo-inositol levels can be monitored as needed while the subject spends everyday life as usual without glucose load. For example, it is possible to grasp the degree of impaired glucose tolerance or the degree of insulin secretory defect using the maximum myo-inositol level or the difference between the maximum myo-inositol level and the minimum myo-inositol level in a day. In addition, monitoring urinary myo-inositol levels as needed allows the subject to reconsider the diet contents and control the amount of exercise to prevent diabetes or the progress thereof while having a regular life.

The methods of monitoring urinary myo-inositol levels include any types of methods capable of detecting myo-inositol, for example, a method using a test paper onto which an enzyme that acts on myo-inositol is fixed and a method of electrochemically detecting myo-inositol using an electrode as a sensor onto which an enzyme acting on myo-inositol is fixed.

In the test paper method, for example, hydrogen peroxide is generated by oxidase and reacted to peroxidase to generate active oxygen, and the active oxygen causes the oxidation of chromogen for coloration, the intensity of which may be observed. The chromogen includes, but not limited to, potassium iodide, tetramethylbenzidine, N-(3-sulfopropyl)-3,3',5,5-'-sodium tetramethylbenzidine, 4-aminoantipyrine, and O-tolidine.

For detecting by means of the sensor, for example, when oxidase is used, hydrogen peroxide generated may be directly measured using an electrode; or the oxidation-reduction current obtained through an electron carrier such as a ferrocene derivative or a quinone derivative or the quantity of the electric current may be measured Likewise, when dehydrogenase is used, the reduced coenzyme may be directly measured using an electrode; or the oxidation-reduction current obtained through an electron carrier or the quantity of the electric current may be measured. Examples are shown in "Biosensor and Quantitative Assay of Substrate Using the Same (Application No. JP 09-263492)" and the like.

In addition, for example, daily monitoring of urinary myo-inositol levels can be more easily carried out by incorporating the above sensor directly into a toilet stool or the like or into a device attached thereto. Such a device may further have functions of memorizing the measurements and of connecting to a terminal of an information processor. In this way, even when the subject stays in a distant place, a medical practitioner or medical institution can make contact with the subject through an electric medium to manage vital data; to give a medical advice; and to examine the degree of impaired glucose tolerance and the degree of insulin secretory defect, leading to review of the diet contents, control of the amount of exercise, improvement of life style, and the medical treatment.

In the case of making quantitative determination of glucose together with myo-inositol in a sample to detect mild impaired glucose tolerance and/or insulin secretory defect, myo-inositol is quantitatively determined preferably by the method of the present invention, while glucose may be quantitatively determined using any conventional methods.

In addition, more precise management can be performed by combining the results of the determination and a doctor's observation. Furthermore, because it is possible to determine the risk to develop diabetes by finding the precondition to diabetes, i.e. prediabetes, and also mild impaired glucose tolerance and insulin secretory defect, which are not prediabetic at present but are highly likely to change to diabetes or prediabetes in the near future, though such finding being impossible by a conventional marker, for example, the risk can be used as an item of examination for life insurance or the like.

For quantitatively determining myo-inositol in a sample, 1 to 500 .mu.L of the sample is added to the composition for myo-inositol quantitative determination to allow a reaction at 37.degree. C. and then the amounts of a coenzyme changed may be directly or indirectly determined for several minutes or several tens of minutes between two time points after the reaction starts, for example, for 1 minute between 3 minutes and 4 minutes after the reaction initiation or for 5 minutes between 3 minutes and 8 minutes. In this case, the myo-inositol content in the sample can be determined by making a comparison with changes in absorbance which are measured for known concentrations of myo-inositol.

The composition (reagent) for the quantitative determination need to contain at least an enzyme that acts on myo-inositol and preferably it further contains a coenzyme.

In addition, a surfactant such as polyoxyethylene octylphenyl ether (OP-10) may be added to the present reagent as appropriate.

Furthermore, the present reagent is used in a form of a liquid product, a freeze-dried product, or a frozen product.

For quantitatively determining myo-inositol in a sample, any types of methods using an enzyme to quantitatively determine myo-inositol may be used. The enzyme to be used in the present invention, which is capable of quantitatively determining myo-inositol, includes any enzymes that act on at least myo-inositol. Of those, however, myo-inositol dehydrogenase is preferable, and myo-inositol dehydrogenase derived from Flavobaterium sp. 671 (FERM BP-7323, hereinafter abbreviated as F.sp.671) is most preferable. In addition, preferably, the myo-inositol dehydrogenase to be used has as low as possible or no contamination of substances that adversely affects coenzymes such as thio-NAD and NADH in the reagent, for example, substances having the activity of decomposing coenzymes, such as NADH oxidase.

The strain F.sp.671 is deposited on an international basis with the deposit number of FERM BP-7323 (date of deposit: Oct. 12, 2000) at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, The Ministry of International Trade and Industry, located at 1-1-3 Higashi, Tsukuba, Ibaraki, Japan (at present: International Patent Organism Depositary, the National Institute of Advanced Industrial Science and Technology, Independent Administrative Agency, located at Center 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan).

For the detection of myo-inositol, any types of methods capable of detecting myo-inositol may be used. The methods include: a method using a visible light coloring reagent, for example, typically yellow coloring with thio-NAD, blue coloring with nitro blue tetrazolium (NBT), or red coloring with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT); a luminescence method; a fluorescence method; a method involving detection of an electric change; and a combination of these methods with amplification techniques.

In addition, using a compact device which can utilize any one of the above methods, the determination of urinary myo-inositol can be carried out non-invasively without restriction on time and place.

The assay for determining the activity of myo-inositol dehydrogenase is as follows:

(1) Activity Assay

<Composition of Reaction Solution> 100 mM Tris buffer (pH 8.5) 20 mM myo-inositol (Sigma Co., Ltd.) 2 mM nicotinamide adenine dinucleotide (NAD) (Oriental Yeast Co., Ltd.) 5 U/ml diaphorase (Asahi Kasei Corporation) 0.025% nitro blue tetrazolium (NBT; Wako Pure Chemical Industries, Ltd.) 1.5% Triton-X100 (Wako Pure Chemical Industries, Ltd.)

One ml of the above reaction solution is added to a small test tube. After the reaction solution is incubated at 37.degree. C. for 5 minutes, 20 .mu.l of an enzyme solution diluted by B times is added thereto and mixed to start the reaction. After the reaction exactly for 5 minutes, 2 ml of 0.1 N HCl is added and mixed to stop the reaction. An absorbance at 550 nm is measured to obtain A1. In addition, the same reaction solution excluding myo-inositol is used to carry out a similar measurement to obtain the absorbance A0. The enzyme activity can be calculated from the following equation. U/ml=[(A1-A0)/18.3].times.[1/5].times.[3.02/0.02].times.B

Numerals in the equation represent the following meanings. 18.3: Molar absorption coefficient of NTB 5: Reaction time 3.02: Total volume of reaction solution 0.02: Volume of enzyme solution B: Dilution factor of enzyme solution

The properties of myo-inositol dehydrogenase derived from the strain F.sp.671 are as follows:

(2) Enzyme Action

This enzyme produces inosose and a reduced coenzyme in the presence of at least myo-inositol and a coenzyme. The coenzyme includes nicotinamide adenine dinucleotides (hereinafter abbreviated as NADs) such as nicotinamide adenine dinucleotide (NAD), acetylpyridine adenine dinucleotide (acetyl-NAD), nicotinamide hypoxanthine dinucleotide (deamino-NAD), pyridine aldehyde adenine dinucleotide (aldehyde-NAD), nicotinamide adenine dinucleotide phosphate (NADP), thio-nicotinamide adenine dinucleotide (thio-NAD), and thio-nicotinamide adenine dinucleotide phosphate (thio-NADP).

Table 1 (see Original Patent) shows the ratio of relative activities on use of each coenzyme (as 100% when NAD is used as a coenzyme). The relative activities were determined with the coenzyme changed according to the following method.

Relative Activity Assay

<Composition of Reaction Solution> Buffer: 100 mM glycine buffer (pH 10.0) Substrate: 20 mM myo-inositol (Sigma, Co., Ltd.) Coenzyme: 2 mM (NAD, thio-NAD, NADP, thio NADP; Oriental Yeast Co., Ltd.)

One ml of the above reaction solution is added to a quartz cell. Then, the quartz cell is placed in a spectrophotometer adjusted at a temperature of 37.degree. C. The cell is incubated for 5 minutes or more and then 20 .mu.l of an enzyme solution of about 1.0 U/ml is added thereto and mixed. The initial velocity is obtained from an absorbance change per minute at a wavelength peculiar to each reduced coenzyme. The initial velocity obtained with each coenzyme is compared with the initial velocity (100%) obtained using NAD as a coenzyme to provide the relative activity.

(3) Substrate Specificity

According to the relative activity assay described above, the measurement was performed using the same concentration of D-chiro-inositol, D-mannose, D-fructose, D-galactose, mannitol, epi-inositol, or scyllo-inositol in place of the substrate in the reaction solution. Table 2 (see Original Patent) shows the enzyme activity for each substrate referring to the initial velocity of the reaction to myo-inositol as 100%. It is revealed that the enzyme derived from the strain F.sp.671 is dehydrogenase having high specificity to myo-inositol.

The substrates used include D-mannose, D-fructose, D-galactose, mannitol, D-chiro-inositol (as above: Wako Pure Chemical Industries, Ltd.), myo-inositol, epi-inositol, and scyllo-inositol (as above: Sigma, Co., Ltd.).

(4) Optimum pH

Following the relative activity assay described above, the measurement was performed using each of 100 mM tris buffer (pH 7.0-9.0) and 100 mM glycine buffer (pH 9.0-11.0) in place of 100 mM of pH 10.0 glycine buffer in the reaction solution. The measurement showed that the optimum pH was about 11.0 (substrate: myo-inositol).

(5) Molecular Weight

Used were TSK gel G300SW (0.75 .PHI..times.600 mm), eluent: 50 mM phosphate buffer (pH 7.5) +0.2 M Na.sub.2SO.sub.4+0.05% NaN.sub.3, and a molecular marker set of Oriental Yeast Co., Ltd. (Japan), a chromatography apparatus made by Shimadzu Corporation (Japan). For the detection, the absorbance at UV 280 nm and the activity of each fraction were measured. myo-Inositol was used as a substrate in the activity measurement, revealing the molecular weight of 40,000.+-.10,000.

(6) Heat Stability

The enzyme showed almost 100% remaining activity after treatment at 40.degree. C. for 15 minutes. The enzyme solution of about 5 U/ml was subjected to heat treatment for 15 minutes. The remaining activity was measured using the enzyme activity assay described above. In the activity measurement, myo-inositol was used as a substrate.

(7) Km value

Using the relative activity assay described above, the concentration of myo-inositol and the concentrations of NAD and thio-NAD were changed to determine Km values respectively. Using the activity assay described above, the substrate concentration was changed to calculate the Km value.

Km value for substrate myo-Inositol: 1.7.+-.0.2 mM

Km value for coenzyme NAD: 0.04.+-.0.01 mM Thio NAD: 4.5.+-.1 mM

For the quantitative determination of myo-inositol with higher sensitivity, the enzymatic cycling method can be used. An example of the enzymatic cycling method is illustrated in the following equation -- see Original Patent.

For the solution composition of the quantitative reaction of myo-inositol using the enzymatic cycling, two or more of coenzymes are appropriately selected in view of Km values of respective coenzymes of myo-inositol dehydrogenase, and the like, and subsequently the pH condition is adjusted between the optimal pH values of forward reaction/reverse reaction to make an efficient progress in the enzymatic cycling. The amounts of A1 and B 1 should be excess over the myo-inositol content in a sample and also excess over Km values of myo-inositol dehydrogenase for A1 and B1.

When using, for instance, myo-inositol dehydrogenase derived from F.sp.671, the Km values for NAD and thio-NAD are 0.04 mM and 4.5 mM, respectively. For the cycling reaction, thio-NAD and NADH may be selected as coenzymes. The concentrations of A1 and B1 are preferably 0.02 mM to 2 M, particularly preferably 0.05 to 100 mM. The amount of myo-inositol dehydrogenase is preferably 1 to 1000 U/mL, particularly preferably 1 to 100 U/mL. The amounts can be suitably selected on the basis of type and amount of the test sample, the myo-inositol content in the sample to be assayed, and the like; but other amounts may be also allowed.

When hexokinase is used as an enzyme for eliminating sugars presented in a sample, any hexokinase capable of catalyzing the reaction from glucose to glucose-6-phosphate may be used, including hexokinase derived from Bacillus sp. Preferable hexokinase is one having excellent heat stability. The hexokinase having excellent heat stability can be obtained by the method described in "Stable Hexokinase and Production Method Thereof" (JP 2000-078982 A).

Because ADP generated together with glucose-6-phosphate has some inhibitory effect on the reaction in the enzymatic cycling method, the present inventors successfully have used ADP-dependent hexokinase simultaneously with hexokinase to improve substantially the elimination of glucose without any influence on the reaction of myo-inositol dehydrogenase. Glc+ATP+Mg.sup.2+.fwdarw.G-6-P+ADP Glc+ADP+Mg.sup.2+.fwdarw.G-6-P+AMP ATP: Adenosine-5'-triphosphate ADP: Adenosine-5'-diphosphate AMP: Adenosine-5'-monophosphate

The assay of hexokinase activity is conducted as follows.

<Composition of Reaction Solution> 50 mM Tris buffer (pH 8.5) (Sigma, Co., Ltd.) 20 mM glucose (Wako Pure Chemical Industries, Ltd.) 4 mM ATP (Oriental Yeast Co., Ltd.) 5 U/mL glucose-6-phosphate dehydrogenase (Toyobo Co., Ltd.) 1 mM NADP (Oriental Yeast Co., Ltd.) 10 mM magnesium chloride (Wako Pure Chemical Industries, Ltd.)

Solution for dissolving and diluting the enzyme: 50 mM Tris buffer (pH 8.5)

One mL of the above reaction solution is added to a quartz cell with 1-cm optical path length, and incubated at 37.degree. C. for 5 minutes. Then, 20 .mu.L of the enzyme solution, which is diluted B times, is added thereto and mixed to start the reaction. The absorbance at 340 nm is measured from the initiation of the reaction to obtain the absorbance change A1 per minute, which shows a linear reaction. A blind test is also conducted in a similar reaction to obtain the absorbance change A0 per minute, except that 50 .mu.L of the solution for dissolving and diluting the enzyme is added instead of the enzyme solution. The enzyme activity is calculated from the following equation. U/ml=[(A1-A0)/6.22].times.[1.02/0.02].times.B

Numerals in the equation represent the following meanings. 6.22: Millimolar extinction coefficient of NADPH at 340 nm 1.02: Total volume of reaction solution (mL) 0.02: Volume of enzyme solution used in the reaction (mL) B: Dilution factor of enzyme solution

The assay of ADP-dependent hexokinase activity is conducted as follows.

<Composition of Reaction Solution> 50 mM Tris buffer (pH 7.5) 20 mM glucose solution (Wako Pure Chemical Industries, Ltd) 2 mM ADP solution (pH 7.0) (Oriental Yeast Co., Ltd.) 5 U/mL glucose-6-phosphate dehydrogenase (Asahi Kasei Corporation) 1 mM NADP solution (Oriental Yeast Co., Ltd) 2 mM magnesium chloride solution (Wako Pure Chemical Industries, Ltd)

Solution for dissolving and diluting the enzyme: 10 mM Tris buffer (pH 7.5)

Three mL of the above reaction solution is added to a small test tube, and incubated at 37.degree. C. for 5 minutes. Then, 50 .mu.L of the enzyme solution, which is diluted by B times, is added thereto and mixed to start the reaction. The absorbance at 340 nm is measured from the initiation of the reaction to obtain the absorbance change A1 per minute, which shows a linear reaction. A blind test is also conducted in a similar reaction to obtain the absorbance change A0 per minute, except that 50 .mu.L of the solution for dissolving and diluting the enzyme is added instead of the enzyme solution. The enzyme activity is calculated from the following equation. U/ml=[(A1-A0)/6.22].times.[3.05/0.05].times.B

Numerals in the equation represent the following meanings. 6.22: Millimolar extinction coefficient of NADPH at 340 nm 3.05: Total volume of reaction solution (mL) 0.05: Volume of enzyme solution used in the reaction (mL) B: Dilution factor of enzyme solution

The amount of hexokinase is preferably 1 to 1,000 u/mL, particularly preferably 1 to 100 u/mL. The amount of ADP-dependent hexokinase is preferably 1 to 1,000 u/ml, particularly preferably 1 to 100 u/mL. The amounts can be appropriately selected depending on type and amount of the test sample, and other amounts can be also used.

In addition, for the determination of urinary myo-inositol over a wide range of its concentration with good reproducibility, an enzymatic cycling reaction should be effectively performed. As a result of intensive examination on concentrations and ratio of thio-NAD and NADH, two coenzymes to be used in the enzymatic cycling reaction, the present inventors have found that the thio-NAD level is preferably 0.01 mM or more, particularly preferably 2 to 10 mM in a final concentration and the ratio of NADH/thio-NAD is preferably 0.01 to 0.5, particularly preferably 0.01 to 0.1. However, the amounts can be appropriately selected according to type and amount of the test sample, and other amounts may be applied.
 

Claim 1 of 12 Claims

1. A method of detecting mild impaired glucose tolerance or an insulin secretory defect in a subject, wherein the method comprises: providing urine samples from said subject, wherein the samples are obtained before and after glucose load, or before and after a meal; quantitatively determining the myo-inositol level in the samples; and determining that the subject has mild impaired glucose tolerance or that the subject has an insulin secretory defect based on the concentration of myo-inositol in the samples, wherein the increment of the concentration of myo-inositol at a characteristic value or higher than a characteristic value of 0 to 20 .mu.g myo-inositol per mg creatinine when measured as an increasing amount of myo-inositol excreted in the urine in a period from 0.5 to 6 hours after 75 g glucose load indicates the subject has mild impaired glucose tolerance or the subject has an insulin secretory defect.

 

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